Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins

Food Chemistry 194 (2016) 272–278

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Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins Li Wang a,1, Yuan Xu a,1, Sumei Zhou b,1, Haifeng Qian a,⇑, Hui Zhang a, Xiguang Qi a, Meihua Fan c a

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China c Jiangsu Institute of Poultry Sciences, Yangzhou 225125, China b

a r t i c l e

i n f o

Article history: Received 14 April 2015 Received in revised form 1 August 2015 Accepted 2 August 2015 Available online 4 August 2015 Keywords: Vaccinium bracteatum Thunb. Rice protein Surface hydrophobicity Secondary structure Hydrophobic interaction Hydrogen bonding

a b s t r a c t In this study, we investigated the interaction of Vaccinium bracteatum Thunb. leaf (VBTL) pigment and rice proteins. In the presence of rice protein, VBTL pigment antioxidant activity and free polyphenol content decreased by 67.19% and 68.11%, respectively, and L* of the protein–pigment complex decreased significantly over time. L* values of albumin, globulin and glutelin during 60-min pigment exposure decreased by 55.00, 57.14, and 54.30%, respectively, indicating that these proteins had bound to the pigment. A significant difference in protein surface hydrophobicity was observed between rice proteins and pigment–protein complexes, indicating that hydrophobic interaction is a major binding mechanism between VBTL pigment and rice proteins. A significant difference in secondary structures between proteins and protein–pigment complexes was also uncovered, indicating that hydrogen bonding may be another mode of interaction between VBTL pigment and rice proteins. Our results indicate that VBTL pigment can stain rice proteins with hydrophobic and hydrogen interactions. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The genus Vaccinium is widely distributed. Its members are found primarily in the Northern hemisphere, especially in mountainous tropical Asia and Central and South America (Fang & Peter, 2015; Xie, 2005). Many Vaccinium species, such as blueberry, cranberry and lingonberry, have been extensively investigated (McKay, Chen, Zampariello, & Blumberg, 2015; RodriguezMateos, Cifuentes-Gomez, George, & Spencer, 2013; Viljanen, Heiniö, Juvonen, Kössö, & Puupponen-Pimiä, 2014). A number of anthocyanins, polyphenols and phenolic acids have been identified in these plants. These compounds are generally recognized for their potential biological activities, which include induction of apoptosis, inhibition of breast cancer and metastasis, postponement of aging, and regulation of blood glucose and lipids (Mahbub et al., 2013; Prior et al., 2010; Rendeiro et al., 2012). Pigments in Vaccinium plants have also been thoroughly investigated (Lee, Finn, & Wrolstad, 2004; Moyer, Hummer, Finn, Frei, & Wrolstad, 2002), thereby revealing some of their properties and ⇑ Corresponding author at: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. E-mail address: [email protected] (H. Qian). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.foodchem.2015.08.006 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

mechanisms of biosynthesis and staining (Kamiya, Yanase, & Nakatsuka, 2014). Vaccinium bracteatum Thunb., known as Wu Fan Shu or Nan Zhu in traditional Chinese medicine, is distributed throughout China. Extensive information about V. bracteatum has recently been published in China, including its components (Chen & Zhang, 2014), functions (Wang, Zhang, Zhang, Yao, & Zhang, 2010) and utilization (Han et al., 2013). Although the species is still underused, V. bracteatum leaf (VBTL) pigment is used to dye rice in parts of eastern coastal China (Xu, Wang, Li, & Chen, 2013). While pigments of Vaccinium plants have been extensively studied, only preliminary research has been conducted on VBTL pigment, including its extraction (Wang, Jiang, Zhang, & Yao, 2008), purification (Langhansova, Landa, Marsik, & Vanek, 2012; Wang & Yao, 2006), physical and chemical properties (Wang, Xu, & Yao, 2011) and biological activities (Wang et al., 2010, 2013). Researchers have used VBTL pigment to dye white hair, fresh eggs and rice (Hu, Jiang, & Zhang, 2001; Jiang & Chen, 1999). Polyphenols have been found to be the main components responsible for the staining activity (Wei, Liu, Xu, He, & Zhang, 2007; Yu, Chen, & Pang, 2007), but the underlying mechanism is still unclear and the pigment is consequently underutilized. Although some studies have addressed the interaction between polyphenols and proteins (Hudson, Ecroyd, Dehle, Musgrave, & Carver, 2009; Kanakis et al., 2011), the underlying binding

L. Wang et al. / Food Chemistry 194 (2016) 272–278

mechanisms have not been elucidated. This information is accordingly also lacking for the polyphenols of VBTL pigment and rice proteins. In light of the absence of relevant studies, we aimed to investigate the interaction between VBTL pigment and rice proteins using a combination of proteomic and biochemical strategies. We developed a simplified model system in which pigment was incubated with isolated classified rice proteins. We measured changes in VBTL pigment polyphenol content and antioxidant activity during the interaction and applied proteomic strategies, such as surface hydrophobicity and Fourier-transform infrared spectroscopy (FT-IR), to monitor various aspects of the rice proteins. The data acquired in this manner allowed us to characterize interaction patterns between VBTL pigment and rice proteins in detail. 2. Materials and methods 2.1. Reagents and standards Leaves of V. bracteatum were supplied by the Jiangsu Jiujiu Environmental Science and Technology Co. (Wuxi, Jiangsu, China). Rice protein was purchased from King-N Rice Industry Group (Yichun, Jiangxi, China). All other chemicals used in this study were of analytical grade. 2.2. Isolation of rice proteins Protein fractions were extracted according to the Osborne method. Rice protein samples were extracted twice with distilled water for 60 min at 25 °C. After centrifugation of each extract at 3000g for 30 min, the supernatant was used for the determination of the water-soluble protein, albumin. The residue was then extracted successively in a similar manner with 5% NaCl, 70% ethanol and 0.05 M NaOH. The supernatants of each extract were collected separately and used to estimate the salt- (globulin), alcohol- (prolamin) and alkali- (glutelin) soluble fractions. Albumin, globulin and glutelin were concentrated by isoelectric sedimentation at pH 4.1, 4.3 and 4.8, respectively, while prolamin was concentrated by evaporation. Residue remaining after the successive extractions corresponded to the insoluble proteins. Each fraction was collected and lyophilized by a lyophilizer (ACPHA1-4, CHRIST, Germany). The protein content of each fraction was determined by the Kjeldahl method (total nitrogen
5.95). 2.3. Preparation of VBTL pigment VBTLs were dried in a forced-air convention oven at 35 °C for about 24 h until a moisture content below 10% was reached. The leaves were then ground to a 40-mesh power and stored at 4 °C in a refrigerator until analysis. The VBTL powder (100 g) was transferred to a dark-colored flask and mixed with 1000 ml of 40% (v/v) ethanol in a constanttemperature bath at 40 °C for 2 h. The ethanolic extract was then centrifuged at 4 °C and 3000g for 20 min. Afterwards, the supernatant was collected and the residue was re-extracted twice. Combined supernatants were evaporated to dryness in vacuo at 40 °C using a vacuum concentrator and then lyophilized (Alpha 1-4, Christ, Osterode, Germany). The crude VBTL pigment was stored at 4 °C until further purification. Crude VBTL pigments (5 g) was dissolved in 50 ml water and loaded onto a chromatographic column (35
400 mm) packed with AB-8 macroporous resins. The column was washed with distilled water and 20% (v/v) ethanol until the eluent was colorless. The column was then eluted with 60% (v/v) ethanol at a flow rate of 1 ml min1. The eluent was collected and concentrated to

273

completely remove the ethanol and then loaded onto another chromatographic column packed with AB-8 macroporous resins. This column was eluted with ethyl acetate at a flow rate of 1 ml min1. The eluent was collected and concentrated until the ethyl acetate was completely removed and then lyophilized. The VBTL pigment was finally stored at 4 °C for use in staining experiments. Purified VBTL pigments (0.1 g) was dissolved in 50 ml distilled water prior to the addition of 2 g of each rice protein fraction. Pigment and proteins were allowed to interact at 70 °C for 60 min using a lab mixer. The resulting complex was filtered using No. 40 Whatman filter paper and washed with excess distilled water to remove redundant pigment before lyophilization. The freeze-dried complex was stored at 4 °C for further analysis. Rice protein (2 g) was dissolved in 50 ml distilled water and then incubated at 70 °C for 60 min. The resulting protein was treated by the same way as the pigment–protein complex. Measured polyphenol, protein and carbohydrate contents of the lyophilized powder were 61.97, 1.12 and 35.64%, respectively. 2.4. Color measurements Color of powdered proteins and pigment–protein complex was measured with a high-precision color-measuring spectrometer (HunterLab, Reston, Virginia, USA), with the data generally represented by CIE Lab color space system parameters. In this system, each point in three-dimensional Cartesian space represents a different color, with its L*, a* and b* values respectively corresponding to its position along black-to-white, green-to-red and blue-toyellow axes. The distance between two measured points is termed the color aberration, represented as DL*, Da* and Db*. 2.5. Determination of antioxidant activity and free polyphenol content VBTL antioxidant activity was determined by a 2,20 -azino-bis-3ethylbenzthiazoline-6-sulfonic acid assay according to the method of Pellegrini, Del, Colombi, Bianchi, and Brighenti (2003). Results were expressed in terms of Trolox equivalent antioxidant capacity (mmol of Trolox per kg [solid foods and oils] or L [beverages] of sample). Polyphenol content was determined by the Folin– Ciocalteu colorimetric method (Cai et al., 2010), with the results expressed as gallic acid equivalents in mg per g of extract. 2.6. Measurement of protein surface hydrophobicity Protein surface hydrophobicity (S0) was determined using 1-anilino-8-naphthalene sulfonate (ANS) as a hydrophobic probe. The proteins were dispersed into phosphate buffer (pH 7.0, 10 mmol/L) and the isolates were serially diluted with the same buffer in concentrations of 0.0005% to 0.015% (w/v). Ten microliters of ANS (8.0 mM in 0.01 M phosphate buffer, pH 7.0) was added to 2 ml of diluted protein solutions, and the final concentration of ANS was 0.04 mM. Fluorescence intensities (FIs-the height of the emission peak at the wavelength of emission maximum) of ANS–protein conjugates were measured with an AmincoBowman spectrofluorometer (Hitachi, Tokyo, Japan). Excitation and emission wavelengths were 390 nm and 470 nm, respectively. The slopes of the plots of net FI versus percent protein were calculated by least squares linear regression and specified as S0. 2.7. Analysis of secondary structure The secondary structures of pigment–albumin, pigment–globulin, pigment–glutelin, and each protein were analyzed at different staining times (0, 4, 8, 12, 20, 40 and 60 min) by FT-IR following the method of Nabet and Pezolet (1997). Briefly, the solid powder was measured by IS10 fourier transform infrared spectrometer

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(Thermo Fisher Scientific, Waltham, MA, USA). The 1600– 1700 cm1 bands were analyzed using software OMINC according to the order of smoothing and deconvolution. The peak position and peak width were recorded. Then the FT-IR figures were analyzed using software Peakfit (V4.12). The corresponding peaks were added in the figure and fitted. At last, bands corresponding to different structures were found and the contents of secondary structures were calculated according to references (Nabet & Pezolet, 1997). The analysis was carried out using OMINC and PeakFit (v4.12) software. 2.8. Statistical analysis Results were expressed as means ± SD. Data were analyzed by one-way analysis of variance followed by a Student–Newman– Keuls post hoc test. Differences among means were considered statistically significant at p < 0.05. 3. Results and discussion Before investigating the interaction between VBTL pigment and rice protein, we analyzed the composition of the latter. The concentrations of albumin, globulin, prolamin, and glutelin were 5.79, 4.13, 5.83, and 60.92%, respectively, and their purities were 68.72, 82.94, 15.82, and 95.26%, respectively. The content of glutelin was significantly higher than that of the other proteins, while the purity of prolamin was significantly lower. On the basis of

18

these values, we chose three types of proteins—albumin, globulin and glutelin—for use in subsequent staining experiments. 3.1. Total antioxidant activity and polyphenol content of pigment and pigment–protein complexes Antioxidant activities and free polyphenol contents of VBTL pigment and pigment–protein complexes are shown in Figs. 1a and b, respectively. We observed a positive correlation between antioxidant activity and free polyphenol content. Polyphenols such as oligomeric proanthocyanidins can increase antioxidant activity because of their slow solubilization and conversion to large polymers over the course of an experiment (Cheynier, 2005). We observed only slight changes in antioxidant activity and free polyphenol content of VBTL pigment alone, indicating that these two parameters were unaffected by the experimental reaction conditions. By the end of the experiment, antioxidant activity and free polyphenol content of the pigment–total protein complex had decreased by 67.19% and 68.11%, respectively. We hypothesize that a large proportion of the free polyphenols combined with the rice protein, thereby leading to the weaker antioxidant activity. With respect to the three systems incorporating different proteins (albumin, globulin and glutelin), we also observed significant decreases in antioxidant activity and free polyphenol content between 0 min and 60 min (Figs. 1a and b). This result demonstrates the dramatic effect of added protein on pigment antioxidant activity and polyphenol content, similar to the findings of a

a

0 min 4 min 8 min 20 min 60 min

16

mM trolox/L

14 12 10 -46.48%

8 -60.56% -67.19%

6

-68.83%

4 2 0 pigment

mg Galllc Acid equivalents/ml

4.0

pigment-total protein albumin+pigment

globulin+pigment

b

glutelin+pigment

0 min 4 min 8 min 20 min 60 min

3.5 3.0 2.5 2.0

-50.30%

1.5

-51.88%

-53.91%

-68.11%

1.0 0.5 0.0 pigment

pigment-total protein pigment-albumin

pigment-globulin

pigment-goltelin

Fig. 1. Antioxidant activity and free polyphenol content of VBTL pigment and pigment–protein complexes during interaction for 0, 4, 8, 20 and 60 min. (a) Antioxidant activity of VBTL pigment and pigment–protein complexes. Values are expressed as mmol Trolox per liter of sample. (b) Free polyphenol content of VBTL pigment and pigment–protein complexes. Values are expressed as gallic acid equivalents in mg per g of sample. Each bar represents means ± SD (n = 3 in each group).

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previous study involving b-lactoglobulin and tea polyphenols (Stojadinovic et al., 2013). Although all three proteins caused decreases in the two measured parameters, the extent of the decreases differed significantly. In particular, antioxidant activity decreased by 46.48, 60.56 and 68.83% in the presence of albumin, globulin and glutelin, respectively, with corresponding decreases in free polyphenol content of 50.30, 51.88 and 53.91%. This suggests that it may be easier for VBTL pigment to react with such protein, which has small solubility and high surface hydrophobicity (Gallo, Vinci, Graziani, De Simone, & Ferranti, 2013; Stojadinovic et al., 2013). We thus speculate that the VBTL pigment combined most easily with the protein having the strongest S0 by hydrophobic modes. 3.2. Surface hydrophobicity of proteins and pigment–protein complexes Measured S0 values of proteins and pigment–protein complexes are shown in Fig. 2. In the protein-only systems, S0 increased during the first 20 min and then decreased slightly. Although several factors were responsible for this increase, it was mainly the result of the exposure of large numbers of hydrophobic groups in the protein molecules upon heating (Wani, Sogi, Singh, & Shivhare, 2011). As the experiment duration was extended beyond 20 min, however, the protein molecules were almost completely relaxed and began to slowly denature as heat was absorbed—in other words, the protein molecules lost their original structures and began to precipitate, causing a slight decrease in S0 (Chen & Zhang, 2014; Wang & Tan, 1997). In the pigment–protein complex systems, S0 showed a similar trend, but with significantly lower values compared with the protein-only systems (Fig. 2). Generally speaking, the differences

between the two systems (Table 1) at each time point may reflect the degree of hydrophobic interaction between VBTL pigment and each rice protein. As shown in Table 1, the differences in S0 between each protein and its corresponding pigment–protein complex were significantly lower (p < 0.05) after the interaction than before. The S0 value of the albumin system (2004.5) was significantly smaller than the values of globulin (7964.5) and glutelin (17172.7) systems. Among the three protein and pigment–protein systems, the smallest difference was observed between albumin and pigment–albumin. For globulin and glutelin, S0 decreased by 40–60% after protein combination with pigment, while the S0 of albumin decreased by 20%. These results indicate that VBTL pigment can most easily interact with globulin and glutelin hydrophobically. Polyphenols have been reported to interact in this manner with proteins such as human blood serum protein (Zou & Xie, 2013). Another study has revealed that polyphenols can combine with hydrophobic groups located on the inside of the protein as well as the surface (Shi, He, & Haslam, 1994). Because its main components are polyphenols, VBTL pigment might combine with proteins in the same fashion. With surface hydrophobic groups exposed to water while other hydrophobic groups are inside, pigment polyphenols can be absorbed both on the protein surface or on interior hydrophobic groups. This dual capability suggests that hydrophobic interaction is one of the main modes of combination between VBTL pigment and rice proteins. 3.3. Color of proteins and pigment–protein complexes Colors of proteins and protein–pigment complexes at different times are shown in Table 2. No significant color changes were observed in the three proteins, but protein–pigment complex colors changed significantly. In particular, L* values of B

a

10000

b

24000

A

8000

surface hydrophobicity

surface hydrophobicity

20000

B

6000

4000

A 16000

12000

B 8000

4000

2000

0 0

10

20

30

40

50

60

0

10

20

t/min

30

40

50

60

t/min

B

c

35000

surface hydrophobicity

30000

A

25000 20000 15000 10000

B 5000 0 0

10

20

30

40

50

60

t/min

Fig. 2. Surface hydrophobicity of proteins and their corresponding protein–pigment complexes. (a) Surface hydrophobicity of albumin (A) and the pigment–albumin complex (B). (b) Surface hydrophobicity of globulin (A) and the pigment–globulin complex (B). (c) Surface hydrophobicity of glutelin (A) and the pigment–glutelin complex (B). Data points represent means ± SD (n = 3 in each group). Points at different times are significantly different (p < 0.05).

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Table 1 Differences in surface hydrophobicity of proteins and pigment–protein complexes. 4 min Albumin Globulin Glutelin

8 min a

1136.0 ± 110.2 2761.5 ± 36.0a 12932.6 ± 1498.5a

12 min b

515.9 ± 169.2 3410.8 ± 1636.3a 17666.7 ± 2190.5b

20 min c

40 min b

1566.0 ± 181.2 4891.0 ± 817.6b 15328.9 ± 211.6a

1190.4 ± 153.1 5418.2 ± 217.0b 17112.3 ± 282.0b

60 min d

2004.5 ± 271.9d 7964.5 ± 674.6c 17172.7 ± 1914.4b

2013.9 ± 114.2 8684.3 ± 205.1c 17064.6 ± 861.2b

Data are expressed as means ± SD (n = 3 in each group). Values in the same row with different superscript letters are significantly different (p < 0.05).

Table 2 Color of proteins and protein–pigment complexes at different interaction times. Protein

Time (min)

L*

a*

Protein

Protein–pigment NS

b*

Protein

Protein–pigment NS

Protein

Protein–pigment NS

Albumin

0 4 8 20 60

90.48 ± 1.32 91.29 ± 0.35 92.16 ± 0.42 88.84 ± 0.85 90.23 ± 0.30

— 87.62 ± 0.87c 86.49 ± 2.93c 61.85 ± 2.31b 40.72 ± 0.76a

1.91 ± 0.13 1.98 ± 0.06 1.95 ± 0.01 1.90 ± 0.03 2.01 ± 0.03

— 2.32 ± 0.19NS 2.63 ± 0.01 2.71 ± 0.13 2.37 ± 0.21

3.47 ± 0.01 3.55 ± 0.37 3.46 ± 0.21 3.41 ± 0.01 3.57 ± 0.14

— 13.8 ± 0.73c 13.4 ± 1.45c 8.17 ± 0.04b 3.32 ± 0.01a

Globulin

0 4 8 20 60

82.15 ± 0.29NS 82.54 ± 0.11 82.50 ± 0.72 83.25 ± 0.01 82.15 ± 0.01

— 78.80 ± 0.75d 75.21 ± 1.51c 62.31 ± 0.08b 35.21 ± 1.48a

0.26 ± 1.63NS 1.54 ± 0.40 0.97 ± 0.01 0.91 ± 0.01 0.78 ± 0.21

— 0.04 ± 1.95a 0.16 ± 1.48a 1.10 ± 0.01b 0.48 ± 0.10a

2.40 ± 1.64NS 3.56 ± 0.98 3.39 ± 0.09 2.51 ± 1.48 2.46 ± 0.39

— 10.14 ± 0.45c 9.86 ± 0.55c 4.36 ± 0.17b 3.05 ± 0.74a

Glutelin

0 4 8 20 60

83.69 ± 0.65NS 82.26 ± 1.37 84.42 ± 2.88 80.80 ± 0.66 82.14 ± 1.01

— 75.42 ± 1.19d 67.86 ± 0.84c 64.17 ± 0.54b 38.25 ± 2.76a

1.15 ± 3.40NS 0.44 ± 1.88 1.61 ± 2.60 0.84 ± 0.01 3.76 ± 0.72

— 2.64 ± 1.72b 1.72 ± 1.06a 1.49 ± 2.51b 1.87 ± 0.02b

0.72 ± 0.36NS 1.17 ± 0.08 1.59 ± 0.86 1.04 ± 0.09 1.05 ± 0.13

— 13.16 ± 0.43d 10.87 ± 0.55c 8.21 ± 0.06b 3.94 ± 0.26a

Data are expressed as means ± SD (n = 3). Values in the same row with different superscript letters are significantly different (p < 0.05); NS indicates values in the same row are not significantly different.

protein–pigment complexes decreased significantly over time. In complexes with the three proteins albumin, globulin and glutelin, L* decreased by 55, 57 and 54%, respectively. These decreases indicate that the protein had combined with the VBTL pigment within 60 min. Correlations between color aberration (DL*, Da* and Db*) and protein hydrophobicity difference (DS0) are shown in Table 3 for proteins and protein–pigment complexes. We previously speculated that the proteins interacted with VBTL pigment over time via hydrophobic bonding. As shown in Table 3, significant positive correlations were uncovered between DL* and Db* vs. DS0 of each protein (Pearson’s r = 0.752–0.960), which supports our hypothesis. Among the three proteins, the correlation coefficient for albumin (0.814) was lower than coefficients for glutelin and globulin (0.896 and 0.931, respectively), implying that glutelin and globulin colors were more strongly related to S0. We propose

Table 3 Correlations between color aberration and surface hydrophobicity difference in proteins and protein–pigment complexes.

DL* Da* Db *

Hydrophobicity differences of pigment–albumin complex (DS0)

Hydrophobicity differences of pigment–glutelin complex (DS0)

Hydrophobicity differences of pigment–globulin complex (DS0)

0.814** 0.235 0.802**

0.896** 0.434* 0.752*

0.931** 0.269 0.960**

Correlations were expressed as Pearson product-moment correlation coefficients, which reflect the degree of linear relationship between two variables. An r coefficient of 1 or 1 corresponds to a perfect positive or negative linear relationship between variables. When 0.7 < |r| < 0.99 or 0.4 < |r| < 0.69 or 0.1 < |r| < 0.39, high, moderate or low linear relationships are respectively indicated between variables. * refers to p < 0.05. ** refers to p < 0.01.

that hydrophobic bonding is the main mode of interaction between VBTL pigment and glutelin/globulin, whereas pigment and albumin interact in a different fashion because the correlation coefficient is different. 3.4. Secondary structures of proteins and pigment–protein complexes The relative content of secondary structures of the rice proteins albumin, globulin and glutelin and rice protein–pigment complexes are shown in Table 4. A significant difference was observed in secondary structures between each protein and protein–pigment complex. After staining with VTBL pigment for 60 min, each protein exhibited significant decreases in a-helix and b-turn structures and significant increases in b-sheets and random coils. Heat absorption has been shown to decrease the proportion of a-helix structures and to increase that of random coils while also altering b-sheet and b-turn structures (He, Shao, & Zhang, 2012). Protein–pigment complexes showed a similar trend, except that greater decreases were detected in a-helix and b-turn structures and greater increases in b-sheets and random coils (Table 4). These results are in agreement with those of several previous studies (Hasni et al., 2011; Hudson et al., 2009; Jöbstl, Howse, Fairclough, & Williamson, 2006) in which the interaction between polyphenols and proteins was found to affect protein structures. We found that VBTL pigment had a high polyphenol content, which decreased significantly after interaction with protein for 60 min (Fig. 1). According to previous reports, polyphenols can easily link with proteins by hydrogen bonding, which in turn may disrupt or destroy hydrogen bonding inside proteins and reduce normal protein folding (Gallo et al., 2013; Hasni et al., 2011). As a result, a-helix and b-turn structures decrease and random coils increase significantly. Gallo et al. (2013) hypothesized that a combination of hydrogen bonding and weak non-covalent bonding were the main contributors to the interaction of tea polyphenols and milk

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L. Wang et al. / Food Chemistry 194 (2016) 272–278 Table 4 Relative content of secondary structures corresponding to proteins (P) and pigment–protein (P–P) complexes subjected to identical conditions.

a-Helix

Time (min)

b-Sheet

b-Turn

Random coil

P

P–P

P

P–P

P

P–P

P

P–P

Albumin

0 4 8 20 60

40.62 ± 0.24d 39.27 ± 0.85d* 32.54 ± 0.54c* 28.89 ± 0.12b* 23.51 ± 0.48a*

— 32.08 ± 1.00d 29.41 ± 0.93c 22.00 ± 0.53b 14.30 ± 0.37a

20.47 ± 0.24a 22.77 ± 0.09b* 24.78 ± 0.93c* 28.57 ± 0.75d* 33.95 ± 0.34d

— 28.54 ± 0.10a 30.55 ± 0.34b 30.57 ± 0.28b 33.16 ± 0.29c

28.86 ± 0.24d 26.71 ± 0.95c 26.48 ± 1.00c* 24.99 ± 0.08b 21.27 ± 0.48a

— 25.46 ± 0.06c 23.83 ± 0.11b 24.40 ± 0.29b 20.47 ± 0.45a

8.26 ± 0.08a 9.07 ± 0 .19a 10.92 ± 0.08b* 11.71 ± 0.23b* 16.60 ± 0.49c*

— 9.30 ± 0.08a 15.21 ± 0.24b 19.96 ± 0.18c 22.00 ± 0.15d

Glutelin

0 4 8 20 60

33.67 ± 1.02e 29.06 ± 0.72d* 22.37 ± 0.62c* 13.41 ± 0.94b* 9.97 ± 0.04a

— 17.74 ± 0.49c 16.99 ± 0.24c 11.34 ± 0.21b 9.02 ± 0.94a

21.14 ± 0.42a 24.64 ± 0.68b 26.59 ± 0.12c* 26.62 ± 0.06c* 29.48 ± 0.15d*

— 24.39 ± 0.54a 29.57 ± 0.69b 29.82 ± 0.83b 36.26 ± 0.27c

28.81 ± 1.01d 27.06 ± 0.39c 24.98 ± 0.48b* 24.47 ± 0.72b* 21.07 ± 0.91a*

— 27.57 ± 0.09c 21.26 ± 0.38b 20.27 ± 0.20b 18.07 ± 0.17a

13.87 ± 0.05a 15.04 ± 0.25b* 15.16 ± 0.46b* 17.92 ± 0.21c* 26.46 ± 0.30d*

— 22.03 ± 1.04a 27.84 ± 0.84b 32.47 ± 0.24c 36.76 ± 0.44d

Globulin

0 4 8 20 60

22.07 ± 0.72c 21.26 ± 0.20c* 18.47 ± 0.51b 18.22 ± 0.82b* 11.36 ± 0.19a*

— 19.06 ± 1.04d 17.26 ± 0.75c 15.43 ± 0.35b 10.71 ± 0.07a

36.16 ± 0.48a 37.21 ± 0.29b 37.24 ± 0.31b 39.46 ± 0.17c* 43.87 ± 0.50d

— 36.99 ± 1.03a 37.96 ± 1.01a 42.87 ± 0.46b 45.04 ± 0.92c

30.01 ± 0.36d 29.61 ± 0.50d 27.76 ± 0.78c 23.11 ± 0.28b* 19.45 ± 0.63a*

— 29.64 ± 0.65d 27.08 ± 0.69c 20.44 ± 0.23b 10.46 ± 0.33a

11.24 ± 0.32a 11.79 ± 0.17a* 13.06 ± 0.26b* 16.26 ± 0.54b* 24.68 ± 0.39c*

— 14.67 ± 0.60a 14.72 ± 0.41a 19.58 ± 0.83b 27.26 ± 0.50c

Data are expressed as means ± SD (n = 3). Values in the same column with different superscript letters are significantly different (p < 0.05). Values in the same row with * for each protein and its secondary structure are significantly different (p < 0.05).

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