Food Chemistry 126 (2011) 630639

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Interaction of milk a- and b-caseins with tea polyphenols

Imed Hasni, Philippe Bourassa, Saber Hamdani, Guy Samson, Robert Carpentier, Heidar-Ali Tajmir-Riahi
Dpartement de Chimie-Biologie, Universit du Qubec Trois-Rivires, C.P. 500, Trois-Rivires, Qubec, Canada G9A 5H7

a r t i c l e

i n f o

Article history:
Received 25 June 2010
Received in revised form 27 October 2010
Accepted 12 November 2010

Keywords:
Milk
Tea
Casein
Polyphenol
Secondary structure
FTIR
UVvisible
CD
Fluorescence spectroscopy

a b s t r a c t
The interaction of a- and b-caseins with tea polyphenols (+)-catechin (C), (!)-epicatechin (EC), (!)-epigallocatechin (EGC) and (!)-epigallocatechin gallate (EGCG) was examined at a molecular level, using
FTIR, UVvisible, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on casein stability
and conformation were determined. Structural analysis showed that polyphenols bind casein via both
hydrophilic and hydrophobic interactions with overall binding constants of KCa-cas = 1.8
(0.8) " 103 M!1, KECa-cas = 1.8 (0.6) " 103 M!1, KEGCa-cas = 2.4 (1.1) " 103 M!1 and KEGCGa-cas = 7.4
(0.4) " 103 M!1, KCb-cas = 2.9 (0.3) " 103 M!1, KECb-cas = 2.5 (0.6) " 103 M!1, KEGCb-cas = 3.5
(0.7) " 103 M!1 and KEGCGb-cas = 1.59 (0.2) " 104 M!1. The number of polyphenol bound per protein
molecule (n) was 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) for a-casien and 1.0 (C), 1.0 (EC), 1.1 (EGC)
and 1.5 (EGCG) for b-casein. Structural modelling showed the participation of several amino acid residues
in polyphenolprotein complexation with extended H-bonding network. Casein conformation was
altered by polyphenol with a major reduction of a-helix and b-sheet and increase of random coil and turn
structure suggesting further protein unfolding. These data can be used to explain the mechanism by
which the antioxidant activity of tea compounds is affected by the addition of milk.
! 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Tea is one of the most popular beverages in the world. In recent
years, much has been said about the added health benefit or reduced benefit of adding milk to tea (Ho, Lin, & Shahidi, 2009; Stanner, 2007). The dual effects of milk on the antioxidant capacity of
different tea polyphenols were recently reported (Dubeau, Samson,
& Tajmir-Riahi, 2010). However, the mechanism by which the antioxidant activity of tea polyphenols affected by milk is not yet
known. Caseins are the major phosphoproteins of mammalian milk
and exist as micelles made of polypeptides known as a-, b- and jcaseins (Uversky, 2002). The three casein components are almost
similar in size, molecular weight (24 kDa) and net negative charge
but differ in their degree of unfoldedness (Farrell et al., 2004; Fox &
McSweeny, 1998; Kumosinski, Brown, & Farell, 1993). Caseins belong to the rapidly growing family of unstructured proteins that
have lately attracted much interest due to their unique unfolded
structure under native conditions, brought about by a combination
of high net charge and low intrinsic hydrophobicity (Phadungath,
2005; Uversky, 2002). a-Casein contains two tryptophan (Trp),
Abbreviations: cas, casein; C, catechin; EC, epicatechin; EGC, epigallocatechin;
EGCG, epigallocatechin gallate; FTIR, Fourier transform infrared; CD, circular
dichroism.
Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084.
E-mail address: tajmirri@uqtr.ca (H.-A. Tajmir-Riahi).
0308-8146/$ - see front matter ! 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.11.087

whilst b- and j-caseins have one Trp residue (Kumosinski et al.,

1993). Plant polyphenolic compounds show strong interaction
with globular proteins and can cause protein unfolding. In solution
polyphenols such as catechins (Scheme 1) can form insoluble complexes with milk proteins including b-casein (Liang & Xu, 2003).
The binding affinity of polyphenols to protein is size dependent
and increases with their molecular size (De Fretias & Mateus,
2001). Larger polyphenols like those in black tea are most likely
to form complexes with milk proteins. This binding can affect the
electron donation capacity of catechins by reducing the number
of hydroxyl groups available in the solution. Studies in the past
have shown the effects of milk protein on the antioxidant capacity
of tea polyphenols, whilst the effect of polyphenol complexation
on the stability and conformation of milk proteins has not been addressed (Aguie-Beghin, Sausse, Meudec, Cheynier, & Doullard,
2008; Alexandropoulou, Komaitis, & Kapsokefalou, 2006; Jobstl,
Howse, Fairclough, & Williamson, 2006; Kartsova & Alekseeva,
2008; Shukla, Narayanan, & Zanchi, 2009; Yan, Hu, & Yao, 2009).
Therefore, the structural characterisation of the interaction between milk proteins and polyphenols is a major step in elucidating
the induced effect of polyphenols on milk protein structure and on
the antioxidant activity of tea compounds.
Fluorescence quenching is considered as a useful technique for
measuring binding affinities. Fluorescence quenching is the
decrease of the quantum yield of fluorescence from a fluorophore

I. Hasni et al. / Food Chemistry 126 (2011) 630639

631

Scheme 1. Chemical structure of tea polyphenols.

induced by a variety of molecular interactions with the quencher

molecule (Lakowicz, 1999; Tayeh, Rungassamy, & Albani, 2009).
Therefore, it is possible to use quenching of the intrinsic tryptophan fluorescence of Trp-37, Trp-66 in as1-casein and Trp-193,
Trp-109 in as2-casein as well as Trp-143 of b-casein (Kumosinski
et al., 1993) as a tool to study the interaction of polyphenol with
caseins in an attempt to characterise the nature of tea catechin
casein complexation.
In this contribution, spectroscopic analysis and docking studies
of the interaction of a- and b-caseins with several tea polyphenols
catechin, epicatechin, epigallocatechin and epigallocatechin gallate
(Scheme 1) in aqueous solution under physiological conditions,
using constant protein concentration and various polyphenol contents are presented. Structural analysis regarding catechin binding
mode and the effects of catechincasein complexation on the protein stability and secondary structure is also reported.
2. Materials and methods
2.1. Materials
The compounds a- and b-caseins (with purity of 70% and 98%)
and tea catechins were purchased from SigmaAldrich Chemical
Co. (St. Louis, MO) and used as supplied. Other chemicals were of
reagent grade and used without any further purification.
2.2. Preparation of stock solutions
Casein was dissolved in aqueous solution (8 mg/ml for a-casein
and 11.8 mg/ml for b-casein to obtain 0.5 mM protein content)
containing 10 mM TrisHCl buffer (pH 7.4). Polyphenol (1 mM)
was prepared in TrisHCl and diluted to various concentrations
in TrisHCl (0.5, 0.25 and 0.125 mM). The protein concentration
was determined spectrophotometrically using the extinction coefficients of 11,000 M!1 cm!1 (b-casein, MW = 24,000) and

15,000 M!1 cm!1 (a-casein, MW = 23,600) at 280 nm (Fox &

McSweeny, 1998; Thorn et al., 2005).
2.3. FTIR spectroscopic measurements
Infrared spectra were recorded on a FTIR spectrometer (Impact
420 model, Digilab, Mississauga, ON, Canada), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter,
using AgBr windows. A Solution of polyphenol was added dropwise
to the protein solution with constant stirring to ensure the formation of homogeneous solution and to reach the target polyphenol
concentrations of 0.125, 0.25 and 0.5 mM with a final protein concentration of 0.25 mM. Spectra were collected from hydrated films
after 2 h incubation of casein with polyphenol solution at room
temperature. Interferograms were accumulated over the spectral
range of 4000600 cm!1 with a nominal resolution of 2 cm!1 and
100 scans. The difference spectra [(protein solution + polyphenol
solution) ! (protein solution)] were generated using the water
combination mode around 2300 cm!1 as standard (Dousseau,
Therrien, & Pezolet, 1989). When producing difference spectra, this
band was adjusted to the baseline level in order to normalise the
difference spectra.
2.4. Analysis of protein conformation
Analysis of the secondary structure of caseins and their polyphenol complexes was carried out on the basis of the procedure already reported (Byler & Susi, 1986). The protein secondary
structure was determined from the shape of the amide I band, located at 16601650 cm!1. Fourier self-deconvolution and second
derivative resolution enhancement were applied to increase the
spectral resolution in the region of 17001600 cm!1. The second
derivatives were obtained using a point convolution of 11 or 13.
The resolution enhancement resulting from self-deconvolution
and the second derivative is such that the number and the position
of the bands to be fitted are determined. In order to quantify the

632

I. Hasni et al. / Food Chemistry 126 (2011) 630639

area of the different components of amide I contour revealed by

self-deconvolution and second derivative, a least-square iterative
curve-fitting was used to fit the Gaussian line shapes to the spectra
between 1700 and 1600 cm!1. The details of spectral manipulation
regarding curve-fitting have previously been reported (Beauchemin et al., 2007). The curve-fitting analysis was performed using
the GRAMS/AI Version 7.01 software of the Galactic Industries
Corporation.
2.5. Circular dichroism
CD Spectra of caseins and their polyphenol complexes were recorded with a Jasco J-720 spectropolarimeter (Jasco Inc., Easton,
MA, USA). For measurements in the far-UV region (178260 nm),
a quartz cell with a path length of 0.01 cm was used in nitrogen
atmosphere. Protein concentration was kept constant (12.5 lM),
whilst varying each polyphenol concentration (0.125, 0.25 and
0.5 mM). An accumulation of five scans with a scan speed of
50 nm per minute was performed and data were collected for each
nm from 260 to 180 nm. Sample temperature was maintained at
25 "C using a Neslab RTE-111 (Jasco Inc., Easton, MA, USA) circulating water bath connected to the water-jacketed quartz cuvettes.
Spectra were corrected for buffer signal and conversion to the
Mol CD (De) was performed with the Jasco Standard Analysis software. The protein secondary structure was calculated using
CDSSTR, which calculates the different assignments of secondary
structures by comparison with CD spectra measured from different
proteins for which high quality X-ray diffraction data are available
(Johnson, 1999; Sreerama & Woddy, 2000). The program CDSSTR is
provided in CDPro software package which is available at the website: http://lamar.colostate.edu/~sreeram/CDPro.
2.6. Absorption spectroscopy
The absorption spectra were recorded on a Perkin-Elmer Lambda 40 spectrophotometer (Woodbridge, ON, Canada). Quartz cuvettes of 1 cm were used and the absorption spectra recorded for
free protein (100 lM) and for its polyphenol complexes with each
polyphenol (10200 lM).
The binding constants of the polyphenolcasein complexes
were calculated as reported (Connors, 1987; Stephanos, 1996). It
is assumed that the interaction between the ligand L and the substrate S is 1:1 (mol/mol); for this reason a single complex SL (1:1) is
formed. It was also assumed that the sites (and all the binding
sites) are independent and finally the Beers law is followed by
all species. A wavelength is selected at which the molar absorptivities eS (molar absorptivity of the substrate) and e11 (molar absorptivity of the complex) are different. Then at total concentration St of
the substrate, in the absence of ligand and the light path length is
b = 1 cm, the solution absorbance is

Ao !S bSt

In the presence of ligand at total concentration Lt, the absorbance of a solution containing the same total substrate concentration is

AL !S bS' !L bL' !11 bSL'

where [S] is the concentration of the uncomplexed substrate, [L] the

concentration of the uncomplexed ligand and [SL] is the concentration of the complex) which, combined with the mass balance on S
and L, gives

AL !S bSt !L bLt !11 bSL'

where De11 = e11 ! eS ! eL (eL molar absorptivity of the ligand). By

measuring the solution absorbance against a reference containing

ligand at the same total concentration Lt, the measured absorbance

becomes

A !S bSt D!11 bSL'

Combining Eq. (4) with the stability constant definition K11 = [SL]/
[S][L], gives

DA K 11 D!11 bS'L'

where DA = A ! Ao. From the mass balance expression St = [S] + [SL]

we obtain [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) that
provides the relationship between the observed absorbance change
per centimetre and the system variables and parameters:

DA St K 11 De11 L'

b
1 K 11 L'

Eq. (6) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration.
The double-reciprocal form of plotting the rectangular hyperbola 1y df ) 1x de, is based on the linearisation of Eq. (6) according
to the following equation,

b
1
1

DA St K 11 De11 L' St De11

Thus the double reciprocal plot of 1/DA versus 1/[L] is linear and the
binding constant can be estimated from the following equation

K 11

intercept
slope

2.7. Fluorescence spectroscopy

Fluorometric experiments were carried out on a Perkin-Elmer
LS55 Spectrometer (Woodbridge, ON, Canada). Stock solutions of
polyphenol 1 mM were prepared at room temperature
(24 1 "C). Various solutions of polyphenol (10200 lM) were
prepared from the above stock solutions by successive dilutions
also at 24 1 "C. Solution of casein (100 lV) in 10 mM TrisHCl
(pH 7.4) was also prepared at 24 1 "C. The above solutions were
kept in the dark and used soon after. Samples containing 0.4 ml
of the above casein solution and various polyphenol solutions were
mixed to obtain final polyphenol concentration of 10200 lV with
constant casein content 100 lV. The fluorescence spectra were recorded at kexc = 280 nm and kem from 287 to 500 nm. The intensity
at 350 nm (tryptophan) was used to calculate the binding constant
(K) according to previous literature reports (Bi et al., 2004; Dufour
& Dangles, 2005; He et al., 2005; Tang, Qi, & Chen, 2005).
2.8. Molecular modelling
The docking studies were performed with ArgusLab 4.0.1 software (Mark A. Thompson, Planaria Software LLC, Seattle, WA,
http://www.arguslab.com). The protein structures were obtained
from Kumosinski et al. (1993) and the polyphenol three dimensional structures were generated from PM3 semi-empirical calculations using Chem3D Ultra 6.0. The whole protein was selected
as a potential binding site since no prior knowledge of such site
was available. The docking runs were performed on the ArgusDock
docking engine using regular precision with a maximum of 1000
candidate poses. The conformations were ranked using the Ascore
scoring function, which estimates the free binding energy. Upon
location of the potential binding sites, the docked complex conformations were optimised using a steepest decent algorithm until
convergence, with a maximum of 20 iterations. Amino acid residues within a distance of 3.5 relative to the polyphenol were involved in the complexation.

I. Hasni et al. / Food Chemistry 126 (2011) 630639

3. Results and discussion

3.1. FTIR spectra of caseinpolyphenol complexes
The polyphenolcasein complexation was characterised by
infrared spectroscopy and its derivative methods. Since there was
no major spectral shifting for the casein amide I band at 1656
1652 cm!1 (mainly C@O stretch) and amide II band at 1544
1540 cm!1 (CN stretching coupled with NH bending modes)
(Beauchemin et al., 2007; Krimm & Bandekar, 1986) upon polyphenol interaction, the difference spectra [(protein solution + polyphenol solution) ! (protein solution)] were obtained, in order to
monitor the intensity variations of these vibrations and the results
are shown in Figs. 1 and 2. Similarly, the infrared self-deconvolution with second derivative resolution enhancement and curve-fitting procedures (Byler & Susi, 1986) were used to determine the
protein secondary structures in the presence of polyphenols
(Fig. 3).
At low polyphenol concentration (0.125 mM), an increase in
intensity was observed for the protein amide I at 1656 and amide
II at 1544 cm!1 (a-casein) and 1652 and 1540 cm!1 (b-casein) in

633

the difference spectra of the polyphenolcasein complexes (Figs.

1 and 2, diffs., 0.125 mM). The positive features are located in the
difference spectra for amides I and II bands at 1648, 1540 (Ccasein), 1650, 1542 (ECcasein), 1654, 1541 (EGCcasein) and at 1651,
1542 cm!1 (EGCGcasein) for a-casein polyphenols and at 1645,
1548 (Ccasein), 1655, 1548 (ECcasein), 1652, 1549 (EGCcasein)
and 1647, 1549 cm!1 (EGCGcasein) for b-caseinpolyphenol complexes (Figs. 1 and 2, diffs., 0.125 mM). These positive features are
related to increase of the intensity of the amides I and II bands
upon polyphenol complexation. The increase in intensity of the
amides I and II bands is due to polyphenol binding to protein
C@O, CN and NH groups (hydrophilic interaction). Additional
evidence to support the polyphenol interaction with CN and N
H groups comes from the shifting of the protein amide A band at
3295 cm!1 (NH stretching mode) in the free caseins to 3285
3280 cm!1 upon polyphenol interaction (spectra not shown).
As polyphenol concentration increased to 0.5 mM, a decrease in
the intensity of the amides I and II bands was observed with negative features in the difference spectra for amides I and II bands at
1654, 1530 (Ccasein), 1654, 1530 (ECcasein), 1658, 1538 (EGC
casein) and at 1650, 1537 cm!1 (EGCGcasein) for a-casein and

Fig. 1. FTIR spectra of hydrated films (pH 7.4) in the region of 1800600 cm!1 for free a-casein (0.25 mM), free C (A) (0.5 mM), free EC (B) (0.5 mM), free EGC (C) (0.5 mM) and
free EGCG (D) (0.5 mM) with difference spectra (diff.) of a-caseinpolyphenol complexes (bottom two curves) obtained at different poylphenol concentrations (indicated in
the figure).

634

I. Hasni et al. / Food Chemistry 126 (2011) 630639

Fig. 2. FTIR spectra of hydrated films (pH 7.4) in the region of 1800600 cm!1 for free b-casein (0.25 mM), free C (A) (0.5 mM), free EC (B) (0.5 mM), free EGC (C) (0.5 mM) and
free EGCG (D) (0.5 mM) with difference spectra (diff.) of b-caseinpolyphenol complexes (bottom two curves) obtained at different polymer concentrations (indicated in the
figure).

at 1651, 1539 (Ccasein), 1655, 1539 (ECcasein), 1655, 1538

(EGCcasein) and 1654 and 1532 cm!1 (EGCGcasein) for b-casein
upon polyphenol complexation (Figs. 1 and 2, diffs., 0.5 mM). The
decrease in the intensity of the amide I band at 16561652 cm!1
in the spectra of the polyphenolcasein complexes suggests a major reduction of protein a-helical structure at high polyphenol concentrations. Similar infrared spectral changes were observed for
protein amide I band in several ligandprotein complexes where
major protein conformational changes occurred (Ahmed Ouameur
et al., 2006).
A quantitative analysis of the protein secondary structure for
the free a- and b-caseins and their polyphenol adducts in hydrated films has been carried out and the results are shown in
Fig. 3. The free a-casein has 36% a-helix (1658 cm!1), 20% b-sheet
(1632 and1618 cm!1), 19% turn structure (1675 cm!1), 4% b-antiparallel (1688 cm!1) and random 21% coil (1644 cm!1) (Fig. 3A).
The free b-casein contains 32% a-helix (1658 cm!1), 24% b-sheet
(1632 and 1618 cm!1), 18% turn structure (1674 cm!1), 4% b-antiparallel (1686 cm!1) and random 23% coil (1644 cm!1) (Fig. 3B).
These results are consistent with spectroscopic studies of caseins
previously reported (Curley, Kumosinski, Unruh, & Farrell, 1998;
Malin et al., 2001). Upon polyphenol interaction, a major decrease

of the a-helix and b-sheet occurred, whilst an increase of turn

and random structure was observed in the polyphenolcasein
complexes (Fig. 3A and B). The conformational changes observed
were more pronounced for ECG and EGCG than C and EC complexes. This is indicative of larger perturbations of casein secondary structure by larger and bulkier polyphenols. This is also
consistent with the extra stability of casein complexes with larger
polyphenols, which will be discussed with fluorescence and UV
data further on.
3.2. CD spectroscopy
CD spectroscopy was also used to analyse the protein conformation in the polyphenolcasein complexes. The CD results exhibit
marked similarities with those of the infrared data. The protein
conformational analysis based on CD data suggests that free acasein has a-helical 35%, b-sheet 12%, turn 20% and random coil
33%, whilst free b-casein contains a-helical 33%, b-sheet 16%, turn
17% and random coil 34%. These data for free b-casein are
consistent with the literature report (Chakraborty & Basak, 2007).
Upon polyphenol interaction, major reduction of a-helix was observed from 35% to 33% in free caseins (a and b) to 3224% in

I. Hasni et al. / Food Chemistry 126 (2011) 630639

635

Fig. 3. Second derivative resolution enhancement and curve-fitted amide I region (17001600 cm!1) for free a-casein (A) and free b-casein (B) with their polyphenol
complexes (0.5 mM polyphenol and 0.25 mM protein concentrations at pH 7.4).

the polyphenolcasein complexes. The decrease in a-helix was

accompanied by an increase in the turn and random coil structures.
The major reduction of the a-helix with an increase in the turn and
random structures is consistent with the infrared results that
showed reduction of a-helix with increase in random coil and turn
structure due to a further protein unfolding.

3.3. Hydrophobic interactions

The spectral changes of the casein CH2 antisymmetric and symmetric stretching vibrations, in the region of 30002800 cm!1 were
monitored in order to locate the presence of hydrophobic contact
in the polyphenolcasein complexes. The CH2 bands of the free

636

I. Hasni et al. / Food Chemistry 126 (2011) 630639

a-casein at 2957, 2933, and 2879 cm!1 shifted to 2959, 2935 and

2873 cm (Ccasein), to 2960, 2935 and 2875 cm (ECcasein),

to 2961, 2936, and 2877 cm!1 (EGCcasein) and to 2959, 2938
and 2874 cm!1 (EGCGcasein), upon polyphenol interaction (Figure not shown). Similarly, the CH2 bands of the free b-casein at
2957, 2933, and 2879 cm!1 shifted to 2959, 2934 and 2877 cm!1
(Ccasein), to 2958, 2934 and 2875 cm!1 (ECcasein), to 2959,
2933 and 2874 cm!1 (EGCcasein) and to 2958, 2931 and
2875 cm!1 (EGCGcasein), upon polyphenol interaction (figure
not shown). The shifting of the protein antisymmetric and symmetric CH2 stretching vibrations suggests the presence of hydrophobic interactions via polyphenol rings and hydrophobic
pockets in caseins, which is consistent with fluorescence spectroscopic results discussed below.
!1

!1

3.4. Fluorescence spectra and stability of polyphenolcasein complexes

a-Casein (mixture of as1- and as2-caseins) has two tryptophan

residues Trp-66, and Trp-37 (in as1-casein) and Trp-109 and Trp193 (in as2-casein), whilst b-casein contains one tryptophan Trp143 with intrinsic fluorescence. These tryptophan residues are located in the protein surfaces (Kumosinski et al., 1993). Tryptophan
emission dominates casein fluorescence spectra in the UV region.
When other molecules interact with casein, tryptophan fluorescence may change depending on the impact of such interaction
on the protein conformation (Lakowicz, 1999; Tayeh et al., 2009).
On the assumption that there are (n) substantive binding sites
for quencher (Q) on protein (B), the quenching reaction can be
shown as follows:
nQ B () Q n B

The binding constant (KA), can be calculated as:

K A Q n B'=Q 'n B'

10

where [Q] and [B] are the quencher and protein concentration,
respectively, [QnB] is the concentration of non fluorescent fluorophorequencher complex and [B0] gives total protein concentration:

Q n B' B0 ' ! B'

11

K A B0 ' ! B'=Q'n B'

12

The fluorescence intensity is proportional to the protein concentration as described:

B'=B0 ' / F=F 0

13

Results from fluorescence measurements can be used to estimate

the binding constant of polyphenolprotein complex from Eq. (14):

logF 0 ! F=F' log K A n logQ '

14

The accessible fluorophore fraction (f) can be calculated by the

modified SternVolmer equation:

F 0 =F 0 ! F 1=fKQ ' 1=f

15

where F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting
molecule). K is the SternVolmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional
fluorescence contribution of the total emission for an interaction
with a hydrophobic quencher (Lakowicz, 1999). The plot of F0/
(F0 ! F) vs 1/[Q] yields f!1 as the intercept on y axis and (fK)!1 as
the slope. Thus, the ratio of the ordinate and the slope gives K.
The decrease of fluorescence intensity of casein is monitored at
340 nm for polyphenolcasein systems (Fig. 4AH shows representative results for each system). Representative plots of F0/(F0 ! F) vs

1/[polyphenol] are shown in Fig. 4A0 H0 . Assuming that the observed changes in fluorescence come from the interaction between
polyphenols and casein, the quenching constant can be taken as the
binding constant of the complex formation. The K values given here
are averages of three-replicate runs for polyphenol/casein systems,
each run involving several different concentrations of polyphenol
(Fig. 4). The binding constants obtained were KCa-cas = 1.8 (0.8)
" 103 M!1, KECa-cas = 1.8 (0.6) " 103 M!1, KEGCa-cas = 2.4 (1.1)
" 103 M!1 and KEGCGa-cas = 7.4 (0.4) " 103 M!1, KCb-cas = 2.9
(0.3) " 103 M!1, KECb-cas = 2.5 (0.6) " 103 M!1, KEGCb-cas = 3.5
(0.7) " 103 M!1 and KEGCGb-cas = 1.59 (0.2) " 104 M!1 (Fig. 4A0
H0 ). The binding constants calculated for the polyphenolcasein
suggest low affinity polyphenolcasein interaction compared to
the other strong ligandprotein complexes (Kragh-Hansen, 1990;
Kratochwil, Huber, Muller, Kansy, & Gerber, 2002; Nsoukpo-Kossi
et al., 2007). However, similar binding constants (103 M!1104 M!1)
were also reported for several ligandprotein complexes using fluorescence spectroscopic methods (Bi et al., 2004; Liang, Tajmir-Riahi,
& Subirade, 2008; Sulkowska, 2002). The binding constants of the
larger polyphenolcasein complexes are bigger than those of smaller polyphenolcasein adducts, which can be due to the presence of
more OH groups associated with the bulkier polyphenols. However,
the stability of polyphenols with b-casein is larger than those of the
corresponding a-casein complexes. This can be attributed to the
more hydrophobic nature of b-casein than a-casein due to the presence of five phosphoserine residues in b-casein (Phadungath, 2005).
The number of polyphenol bound per protein (n) is calculated
from log [(F0 ! F)/F] = log KS + n log [polyphenol] for the static
quenching (Charbonneau & Tajmir-Riahi, 2010; Froehlich, Jennings, Sedaghat-Herati, & Tajmir-Riahi, 2009; Jiang, Gao, & He,
2002; Jiang et al., 2004; Liang et al., 2008; Mandeville & TajmirRiahi, 2010). The n values from the slope of the straight line are
for a-casein 1.1 (C), 0.9 (EC), 1.1 (EGC), 1.5 (EGCG) and for b-casein
1.0 (C), 1.0 (EC), 1.1 (EGC) and 1.5 (EGCG).
The f values obtained for polyphenolcasein complexes suggest
that polyphenols interact with fluorophore via hydrophobic interactions. As a result, we predict that polyphenols bind mainly with
the fluorophores located on the surface of caseins. This argument is
based on the fact that the emissions, kmax, of Trp-214 (HSA) and
Trp-212 (BSA) are at 340 nm, which is the emission region of hidden tryptophan molecules, whilst fluorescence emission of exposed tryptophan molecule is at a higher wavelength (350 nm)
due to solvent relaxation (Liang et al., 2008; Sulkowska, 2002; Tayeh et al., 2009). The tightening of protein structure through intramolecular interactions, such as hydrogen bonds, seems to bury
tryptophan in a more hydrophobic environment. The changes in
fluorescence intensity of tryptophan in both a- and b-casein in
the presence of polyphenol exhibited two different patterns. In
the catechin and epicatechin complexes of a- and b-caseins, the
emission band of the free protein at 350 nm (a-casein) and at
348 nm (b-casein) shifted towards a lower wavelength at 346 nm
(Ccasein) and 348 nm (EC) for a-casein, and at 334 nm (Ccasein)
and 343 nm (ECcasein) in b-casein complexes (Fig. 4A, B, E and F).
On the other hand, the emission band of the free caseins shifted towards a higher wavelength at 351 nm (EGCcasein) and 365 nm
(EGCGcasein) for a-casein, and at 349 nm (EGCcasein) and
367 nm (EGCGcasein) in b-casein complexes (Fig. 4C, D, G and
H). The upward shift of the casein emission band was more pronounced in the case of the larger and bulkier EGCG molecule with
more OH groups (Fig. 4D and H). The downward shift of the emission band of casein in the catechin and epicatechinprotein complexes is due to the tightening of protein structure through
intramolecular interactions, such as hydrogen bonds. This seems
to change tryptophan in a more hydrophobic environment. However the upward shift of the protein emission bands observed in
the spectra of EGCcasein and EGCGcasein is related to more

I. Hasni et al. / Food Chemistry 126 (2011) 630639

637

Fig. 4. Fluorescence emission spectra of polyphenolcasein systems in 10 mM TrisHCl buffer pH 7.4 at 25 "C for (A) (Ca-casein) (1) free a-casein 100 lM, (212) C at 10, 20,
40, 60, 80, 100, 120, 140, 160, 180, 200 lM; (B) (ECa-casein) (1) free a-casein 100 lM, (212) EC with similar concentrations as catechin in A; (C) (ECGa-casein) (1) free acasein 100 lM, (212) ECG with similar concentrations as catechin in A; (D) (EGCGa-casein) (1) free a-casein 100 lM, (212) EGCG with similar concentrations as catechin
in A; (E) (Cb-casein) (1) free b-casein 100 lM, (212) C with similar concentrations as catechin in (A); (F) (ECb-casein) (1) free b-casein 100 lM, (212) EC with similar
concentrations as catechin in A; (G) (ECGb-casein) (1) free b-casein 100 lM, (212) ECG with similar concentrations as catechin in A; (H) (EGCGb-casein) (1) free b-casein
100 lM, (212) EGCG with similar concentrations as catechin in A. The plot of F0/(F0 ! F) as a function of 1/polyphenol concentration. The binding constant K being the ratio of
the intercept and the slope for polyphenol complexes with both a- and b-caseins (A0 H0 ).

exposure of tryptophan residue and unfolding of protein structure.

This is consistent with our infrared results that show major secondary structural changes for caseins upon EGC and EGCG
complexation.
3.5. UV spectra and stability of polyphenolcasein complexes
The polyphenolcasein binding constants were also determined
using UVvisible spectroscopic method (described in Section 2).
The double reciprocal plot of 1/DA versus 1/[L] is linear and the
binding constant can be estimated from the intercept to slope.
One binding site was observed for each polyphenolprotein complex with the overall binding constants of KCa-cas = 1.3
(0.2) " 103 M!1, KECa-cas = 1.1 (0.7) " 103 M!1, KEGCa-cas = 2.3

(0.9) " 103 M!1 and KEGCGa-cas = 3.2 (1.4) " 103 M!1, KCb3
!1
, KECb-cas = 2.3 (0.9) " 103 M!1, KEGCbcas = 1.2 (0.2) " 10 M
3
!1
and KEGCGb-cas = 6.1 (4.0) " 103 M!1.
cas = 4.1 (1.2) " 10 M
The association constants calculated for the polyphenolcasein adducts from UV spectroscopy are consistent with those from fluorescence spectroscopy with KCa-cas = 1.8 (0.8) " 103 M!1, KECa3
!1
, KEGCa-cas = 2.4 (1.1) " 103 M!1 and
cas = 1.8 (0.6) " 10 M
KEGCGa-cas = 7.4 (0.4) " 103 M!1, KCb-cas = 2.9 (0.3) " 103 M!1,
KECb-cas = 2.5 (0.6) " 103 M!1, KEGCb-cas = 3.5 (0.7) " 103 M!1
and KEGCGb-cas = 1.59 (0.2) " 104 M!1 (Fig. 4A0 H0 ). The stability
of casein adducts with catechin and epicatechin are very similar
with the same number of OH groups. However, a larger stability
was observed for EGCGcasein than EGCcasein complexes due
to the extra OH groups associated with the bulkier EGCG molecule.

638

I. Hasni et al. / Food Chemistry 126 (2011) 630639

Fig. 5. Best docked conformations of cathechincasein complexes. Residues of interest are shown in red colour and the cathechin in green colour. (A) For epicatechin
complexed to a-casein, (B) for epigallocatechin gallate complexed to a-casein, (C) for epicatechin complexed to b-casein and (D) for epigallocatechin gallate complexed to bcasein.

Table 1
Residues involved in proteincatechin interactions with the free binding energy for the polyphenolcasein complexes.
Complexes (kcal/mol)
ECcasein

EGCGcasein

b
b
a

Residues involved

DGbinding

Gln-30a, Phe-23, Phe-24, Phe-28, Phe-32a, Val-31

Ala-177, Gly-203, Leu-208, Leu-171, Leu-191a, Leu-192, Leu-198, Phe-190, Pro-204, Tyr-193a, Val-178, Val-197a

!9.67
!10.18

Asp-43, Gln-30a, Glu-63a, Glu-77a, His-80a, Ile-65a, Ile-71, Ile-81, Lys-42a, Phe-32, Ser-41
Ala-177, Gly-203, Ile-208, Leu-191, Leu-192a, Leu-198, Phe-190, Pro-179, Pro-204, Tyr-180a, Tyr-193a, Val-178a, Val-197a

!9.29
!11.08

Hydrogen bonding reported with this residue.

3.6. Docking studies

Our results from FTIR, UVvisible, CD and fluorescence spectroscopic methods are accompanied by docking experiments in which
the EC and EGCG molecules were docked to a- and b-caseins to
determine the preferred binding sites on these proteins. The stereoview of the dockings of EC and EGCG are shown in Fig. 5 and Table 1.
The models show that EC is surrounded by Gln-30, Phe-23, Phe-24,
Phe-28, Phe-32 and Val-31, whilst EGCG is in the vicinity of Asp-43,
Gln-30, Glu-63, Glu-77, His-80, Ile-65, Ile-71, Ile-81, Lys-42, Phe-32
and Ser-41 residues in a-casein complexes (Fig. 5 and Table 1). In bcasein complexes, the EC is surrounded by Ala-177, Gly-203, Leu208, Leu-171, Leu-191, Leu-192, Leu-198, Phe-190, Pro-204, Tyr193, Val-178 and Val-197, whilst EGCG is in the vicinity of Ala177, Gly-203, Ile-208, Leu-191, Leu-192, Leu-198, Phe-190, Pro179, Pro-204, Tyr-180, Try-193, Val-178 and Val-197 residues
(Fig. 5 and Table 1). The binding energy (DG) shows more stable
polyphenol complexes formed with b-casein than a-casein, which
is consistent with our spectroscopic data and the binding constants
obtained for the polyphenolcasein complexes.
4. Conclusions
Based on our spectroscopic results and docking studies presented here, tea polyphenols weakly bind to both a-casein and

b-casein in solution. The order of binding increases as the number

of OH group increased with C * EC > EGC > EGCG. b-Casein forms
stronger complexes with tea polyphenols than a-casein, due to
more hydrophobic nature of b-casein. Polyphenolcasein interaction is more hydrophobic than hydrophilic. Polyphenol binding alters casein secondary structure with major decrease in a-helix and
b-sheet accompanied by an increase in random coil and turn structures, leading to more protein unfolding. The casein structural
changes can be a major factor in the effect of milk on the antioxidant activity of tea polyphenols.
Acknowledgments
The financial support of the Natural Sciences and Engineering
Research Council of Canada (NSERC) to H.A. Tajmir-Riahi and R.
Carpentier is highly acknowledged.
References
Aguie-Beghin, V., Sausse, P., Meudec, E., Cheynier, V., & Doullard, R. (2008).
Polyphenolb-casein complexes at air/water interface and in solution: Effects of
polyphenol structure. Journal of Agricultural and Food Chemistry, 56, 96009611.
Ahmed Ouameur, A., Diamantoglou, S., Sedaghat-Herati, M. R., Nafisi, Sh.,
Carpentier, R., & Tajmir-Riahi, H. A. (2006). An overview of drug binding to
human serum albumin: Protein folding and unfolding. Cell Biochemistry and
Biophysics, 45, 203213.

I. Hasni et al. / Food Chemistry 126 (2011) 630639

Alexandropoulou, I., Komaitis, M., & Kapsokefalou, M. (2006). Effects of iron,
ascorbate, meat and casein on the antioxidant capacity of green tea under
conditions of in vitro digestion. Food Chemistry, 94, 359365.
Beauchemin, R., N soukpoe-Kossi, C. N., Thomas, T. J., Thomas, T., Carpentier, R., &
Tajmir-Riahi, H. A. (2007). Polyamine analogues bind human serum albumin.
Biomacromolecules, 8, 31773183.
Bi, S., Ding, L., Tian, Y., Song, D., Zhou, X., Liu, X., et al. (2004). Investigation of the
interaction between flavonoids and human serum albumin. Journal of Molecular
Structure, 703, 3745.
Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by
deconvoluted FTIR spectra. Biopolymers, 25, 469487.
Chakraborty, A., & Basak, S. (2007). Effect of surfactants on casein structure: A
spectroscopic study. Colloids and Surfaces B: Biointerfaces, 63, 8390.
Charbonneau, D., & Tajmir-Riahi, H. A. (2010). Study on the interaction of cationic
lipids with bovine serum albumin. Journal of Physical Chemistry Part B, 114,
11481155.
Connors, K. (1987). Binding constants: The measurement of molecular complex
stability. New York: John Wiley & Sons.
Curley, D. M., Kumosinski, T. F., Unruh, J. J., & Farrell, H. M. Jr., (1998). Changes in the
secondary structure of bovine casein by Fourier transform infrared
spectroscopy: Effects of calcium and temperature. Journal of Dairy Science, 81,
31543162.
De Fretias, N., & Mateus, S. (2001). Structural features of procyanidin interactions
with salivary proteins. Journal of Agricultural and Food Chemistry, 49,
940945.
Dousseau, F., Therrien, M., & Pezolet, M. (1989). On the spectral subtraction of water
from the FT-IR spectra of aqueous solutions of proteins. Applied Spectroscopy, 43,
538542.
Dubeau, S., Samson, G., & Tajmir-Riahi, H. A. (2010). Duel effect of milk on the
antioxidant capacity of green, Darjeeling, and English breakfast teas. Food
Chemistry, 122, 539545.
Dufour, C., & Dangles, O. (2005). Flavnoidserum albumin complexation:
Determination of binding constants and binding sites by fluorescence
spectroscopy. Biochimica and Biophysica Acta, 1721, 164173.
Farrell, H. M., Jr., Jimenez-Flores, R., Bleck, G. T., Brown, E. M., Butler, J. E., Creamer, L.
K., et al. (2004). Nomenclature of proteins of cows milk sixth revisions.
Journal of Dairy Science, 87, 16411674.
Fox, P. F., & McSweeny, P. L. H. (1998). In Dairy chemistry and biochemistry
(pp. 150169). London, UK: Blackie Academic and Professional.
Froehlich, E., Jennings, C. J., Sedaghat-Herati, M. R., & Tajmir-Riahi, Y. F. (2009).
Dendrimers bind human serum albumin. Journal of Physical Chemistry Part B,
113, 69866993.
He, W., Li, Y., Xue, C., Hu, Z., Chen, X., & Sheng, F. (2005). Effect of Chinese medicine
alpinetin on the structure of human serum albumin. Bioorganic & Medicinal
Chemistry, 13, 18371845.
Ho, C.-T., Lin, J.-K., & Shahidi, F. (2009). Tea and tea products: Chemistry and health
promoting properties. Boca Raton, FL: CRC Press.
Jiang, C. Q., Gao, M. X., & He, J. X. (2002). Study of the interaction between terazosin
and serum albumin synchronous fluorescence determination of terazosin.
Analytica Chimica Acta, 452, 185189.
Jiang, M., Xie, M. X., Zheng, D., Liu, Y., Li, X. Y., & Chen, X. (2004). Spectroscopic
studies on the interaction of cinnamic acid and its hydroxyl derivatives with
human serum albumin. Journal of Molecular Structure, 692, 7180.
Jobstl, E., Howse, J. R., Fairclough, J. P. A., & Williamson, M. P. (2006). Noncovalent
cross-linking of casein by epigallocatechin gallate characterized by single
molecule force microscopy. Journal of Agricultural and Food Chemistry, 54,
40774081.
Johnson, W. C. (1999). Analyzing protein circular dichroism spectra for accurate
secondary structure. Proteins: Structure, Function and Genetics, 35, 307312.

639

Kartsova, L. A., & Alekseeva, A. V. (2008). Effect of milk caseins on the concentration
of polyphenolic compounds in tea. Journal of Analytical Chemistry, 63,
11071111.
Kragh-Hansen, U. (1990). Structure and ligand binding properties of human serum
albumin. Danish Medical Bulletin, 37, 5784.
Kratochwil, N. A., Huber, W., Muller, F., Kansy, M., & Gerber, P. R. (2002). Predicting
plasma protein binding of drugs: A new approach. Biochemical Pharmacology,
64, 13551374.
Krimm, S., & Bandekar, J. (1986). Vibrational spectroscopy and conformation of
peptides, polypeptides, and proteins. Advances in Protein Chemistry, 38,
181364.
Kumosinski, T. F., Brown, E. M., & Farell, H. M. Jr., (1993). Three-dimensional
molecular modeling of bovine caseins: A refined, energy-minimized beta-casein
structure. Journal of Dairy Science, 76, 931945.
Lakowicz, J. R. (1999). In Principles of fluorescence spectroscopy (2nd ed.). New York:
Kluwer/Plenum.
Liang, L., Tajmir-Riahi, H. A., & Subirade, M. (2008). Interaction of b-lactoglobulin
with resveratrol and its biological implications. Biomacromolecules, 9, 5055.
Liang, Y., & Xu, Y. (2003). Effect of extraction temperature on cream and
extractibility of black tea [Camellia sinensis (L.) O. Kuntze]. International
Journal of Food Science and Technology, 38, 3745.
Malin, E. L., Alaimo, M. H., Brown, E. M., Aramini, J. M., Germann, M. W., Farrell, H.
M. Jr.,, et al. (2001). Solution structures of casein peptides: NMR, FTIR, CD and
molecular modeling studies of as1-casein, 123. Journal of Protein Chemistry, 20,
391404.
Mandeville, J. S., & Tajmir-Riahi, H. A. (2010). Complexes of dendrimers with bovine
serum albumin. Biomacromolecules, 11, 465472.
Nsoukpo-Kossi, C. N., Sedaghat-Herati, M. R., Ragi, C., Hotchandani, S., & TajmirRiahi, H. A. (2007). Retinol and retinoic acid bind human serum albumin:
Stability and structural features. International Journal of Biological
Macromolecules, 40, 484490.
Phadungath, C. (2005). Casein micelle structure: A concise review. Journal of Science
and Technology, 27, 201212.
Shukla, A., Narayanan, T., & Zanchi, D. (2009). Structure of casein micelles and their
complexation with tannins. Soft Matter, 5, 28842888.
Sreerama, N., & Woddy, R. W. (2000). Estimation of protein secondary structure
from circular dichroism spectra: Comparison of CONTIN, SELCON and CDSSTR
methods with an expanded reference set. Analytical Biochemistry, 287, 252260.
Stanner, S. (2007). Does adding milk remove the benefit of your daily cuppa? British
Nutrition Foundation Nutrition Bulletin, 32, 101103.
Stephanos, J. J. (1996). Drugprotein interactions: Two-site binding of heterocyclic
ligands to a monomeric hemoglobin. Journal of Inorganic Biochemistry, 62,
155169.
Sulkowska, A. (2002). Interaction of drugs with bovine and human serum albumin.
Journal of Molecular Structure, 614, 227232.
Tang, J., Qi, S., & Chen, X. (2005). Spectroscopic studies of the interaction of anticoagulant rodenticide diphacinone with human serum albumin. Journal of
Molecular Structure, 779, 8795.
Tayeh, N., Rungassamy, T., & Albani, J. R. (2009). Fluorescence spectral resolution of
tryptophan residues in bovine and human serum albumins. Journal of
Pharmaceutical and Biomedical Analysis, 50, 107116.
Thorn, D. C., Meehan, S., Sunde, M., Rekas, A., Gras, S. I., MacPhee, C. E., et al. (2005).
Amyloid fibril formation by bovine milk j-casein, and its inhibition by the
molecular chaperones as and b-casein. Biochemistry, 44, 1702717036.
Uversky, V. N. (2002). Natively unfolded proteins: Appoint where biology waits for
physics. Protein Science, 11, 739756.
Yan, Y., Hu, J., & Yao, P. (2009). Effects of casein, ovalbumin, and dextran on the
astringency of tea polyphenols determined by quartz crystal microbalance with
dissipation. Langmuir, 25, 397402.