Protein Phenolic Interaction

Food Research International 51 (2013) 954970
Contents lists available at SciVerse ScienceDirect
Food Research International

journal homepage: www.elsevier.com/locate/foodres
Review
A review on proteinphenolic interactions and associated changes

Tugba Ozdal a, Esra Capanoglu b,, Filiz Altay b
a
b
Department of Food Engineering, Faculty of Engineering and Architecture, Okan University, Tuzla, TR-34959, Istanbul, Turkey
Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Maslak, TR-34469, Istanbul, Turkey
a r t i c l e
i n f o
Article history:
Received 1 August 2012
Accepted 9 February 2013
Keywords:
Proteinphenolic interactions
Proteins
Phenolics
Total antioxidant capacity
Bioavailability
a b s t r a c t
Polyphenols have become an intense focus of research interest due to their health-benecial effects especially in the
treatment and prevention of several chronic diseases. Polyphenols are known to form complexes with proteins leading to changes in the structural, functional and nutritional properties of both compounds. In this review, the effects
of proteinphenolic interactions under various conditions on protein and phenolic compound's structure and functionality are described. The parameters that are dened to affect proteinphenolic interactions are basically temperature, pH, protein type and concentration, and the type and structure of phenolic compounds. Even though the exact
mechanism of how proteins inuence polyphenols is still not yet known, studies on the changes in the structure and
functional properties were investigated. According to these studies, secondary and tertiary structures of the proteins
are changed, and solubility of the protein is decreased whereas its thermal stability might be improved. In addition,
the amount of some amino acids and protein digestibility might be reduced as a result of this interaction. It is also
concluded that proteins signicantly decrease the antioxidant capacity in general, but there are some controversial
results which might be due to the differences in the analytical techniques performed in these studies. Similarly,
different results were obtained in the bioavailability experiments. Factors affecting these results as well as lacking
parts of these studies are discussed in detail in this review. In conclusion, interaction of proteins and phenolic compounds is a complex phenomenon and should be further investigated. On the other hand, optimum conditions
should be studied in detail to improve the food processes and provide maximum benecial health effects to the consumers with optimum nutritional and functional properties.
2013 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Parameters affecting interactions between protein and phenolic compounds
.
2.1.
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Types of proteins and protein concentration . . . . . . . . . . . . .
2.4.
Types and structures of phenolic compounds . . . . . . . . . . . . .
2.5.
Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of proteinphenolic compounds interactions on proteins . . . . . . .
3.1.
Effects on structure . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Effects on functional properties . . . . . . . . . . . . . . . . . . .
3.3.
Effects on nutritional value and digestibility . . . . . . . . . . . . .
Effects of proteinphenolic compounds interactions on phenolic compounds .
4.1.
Effects on total phenolic content (TPC), total avonoid content (TFC) and
4.2.
Effects on the content of individual phenolic compounds . . . . . . .
4.3.
Effects on in-vivo bioavailability . . . . . . . . . . . . . . . . . . .
4.4.
Effects on in-vitro bioavailability . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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antioxidant activity
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Corresponding author at: Istanbul Technical University, Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Maslak, TR-34469, Istanbul, Turkey. Tel.: +90
212 285 7340; fax: +90 212 285 7333.
E-mail address: capanogl@itu.edu.tr (E. Capanoglu).
0963-9969/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.02.009
T. Ozdal et al. / Food Research International 51 (2013) 954970
6.
Conclusions
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References
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Proteins are highly complex polymers, made up of twenty different
amino acids consisting of an -carbon atom covalently attached to a hydrogen atom, an amino group, a carboxyl group, and a side-chain R
group (Fig. 1) (Damodaran, 1996). The differences in structure and
function of proteins arise from the sequence in which the amino acids
are linked together via amide bonds. Proteins are important food components existing mostly in milk, meats (including sh and poultry),
eggs, cereals, legumes and oilseeds. They can form complexes with
other food components including polyphenols leading to changes in
their structural, functional and nutritional properties. A fundamental
understanding of these changes as a result of interactions with phenolic
compounds is essential from the scientic, industrial, and economical
point of view.
Phenolic compounds are chemically structured as a hydroxyl group
bonded to an aromatic ring (Fig. 2). They are secondary metabolites, not
involved in growth and energy metabolism in the body (Harnly,
Bhagwat, & Lin, 2007). There are currently more than 8000 known phenolic compounds identied in fruits, vegetables, seeds, and liquids
(Cuykens & Claeys, 2004; Guo, Kong, & Meydani, 2009). Phenolic compounds can be classied into two groups: basic phenolic compounds
and polyphenols (Vermerris & Nicholson, 2006). Dietary polyphenols
represent the main source of antioxidants for human use (Graf,
Milbury, & Blumberg, 2005). The main classes of polyphenols are dened according to the nature of their carbon skeleton: phenolic acids,
avonoids, and the less common stilbenes and lignans. Phenolic acids
include caffeic acid, ferulic acid and hydrolyzable tannins. Flavonoids
can be divided into several classes according to their degree of oxidation
in the heterocycle (Guo, Kong, & Meydani, 2009). Flavonoids include
avonols (e.g., quercetin and kaempferol, the most ubiquitous avonoids
in foods), avones, isoavones, avanones, anthocyanins, avanols
(catechins-monomers and proanthocyanidin polymers, known as condensed tannins) (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004;
Scalbert & Williamson, 2000).
Polyphenols have become an intense focus of research interest
due to their health-benecial effects especially in the treatment and
prevention of cancer (Chen et al., 2011; Weng & Yen, 2012) and cardiovascular diseases (Kuriyama et al., 2006; Mursu et al., 2008). The
suggested benecial effects include anticarcinogenic (Jeong et al.,
2011; Ogunleye, Xue, & Michels, 2009), antiatherogenic (Liu, Zubik,
Collins, Marko, & Meydani, 2004; Mulvihill & Huff, 2010), antiulcer
(Zakaria et al., 2011), antithrombotic (Han et al., 2012; Tao et al.,
2012), anti-inammatory (Beara et al., 2012; Zimmer et al., 2012),
antiallergenic (Chung & Champagne, 2009; Schmitz-Eiberger &
Blanke, 2012), anticoagulant (Bijak et al., 2011), immune modulating
(Schtz, Sa, With, Graubaum, & Grnwald, 2010), antimicrobial
(Silva, Rodrigues, Feas, & Estevinho, 2012; Xia, Wu, Shi, Yang, &
COOH
H
NH2
R
Fig. 1. The structure of an amino acid.
955
968
968
Zhang, 2011), vasodilatory (Mudnic et al., 2010), and analgesic activities (Santoz, Almeida, Lopez, & Souza, 2010).
Polyphenols can be oxidized by molecular oxygen with side chain
amino groups of peptides at alkaline pH to quinines, leading to the formation of protein cross-links (Damodaran, 1996; Prodpran, Benjakul, &
Phatcharat, 2012). These highly reactive quinines can irreversibly react
with the sulfhydryl and amino groups of proteins. In addition, quinines
can undergo condensation reactions, resulting in the formation of high
molecular weight brown colored pigments named as tannins. Tannins
are highly reactive and can readily combine with SH and amino groups
of proteins. Quinoneamino group reactions are known to decrease the
digestibility and bioavailability of protein-bound lysine and cysteine
(Damodaran, 1996).
The phenolic group is an excellent hydrogen donor that forms hydrogen bonds with the carboxyl group of the protein. For phenolic
compounds to have high protein afnity, they must be small enough
to penetrate inter-brillar regions of protein molecules, but large
enough to crosslink peptide chains at more than one point (Mulaudzi,
Ndhlala, Kulkarni, & Staden, 2012). The molecular explanations of proteinphenolic interaction are given in Fig. 3. The diphenol moiety of a
polyphenol (Almajano, Delgado, & Gordon, 2007) is readily oxidized
to an ortoquinone, either enzymatically as in plant tissues, or by molecular oxygen (Damodaran, 1996; Strauss & Gibson, 2004). The quinine
forms a dimer (Arimboor & Arumughan, 2011) in a side reaction, or reacts with amino or sulfhydryl side chains of polypeptides to form covalent C\N or C\S bonds with the phenolic ring, with regeneration of
hydroquinone. The latter can be reoxized and bind a second polypeptide, resulting in a cross-link (Arts, Haenen, Voss, & Bast, 2001). Otherwise, two quinines, each carrying one chain, can dimerize, producing
a cross-link as well (Arts et al., 2002) (Strauss & Gibson, 2004).
The interactions of phenolic compounds with proteins may lead to
changes in physico-chemical properties of proteins such as solubility,
thermal stability, and digestibility (Labuckas, Maestri, Perell, Martnez,
& Lamarque, 2008; Rawel, Kroll, & Rohn, 2001). Additionally nutritional
properties of proteins may be affected due to the modication of essential amino acids and through the inhibition of proteases (Kroll, Rawel, &
Rohn, 2003). On the other hand, the interactions of other compounds including lipids (Smith, 2012), other proteins (Hsu, Pang, Sheetal, &
Wilkins, 2007; Thangudu, Bryant, Panchenko, & Madej, 2012), vitamins
(Relkin & Shukat, 2012) with proteins are also known to change the
characteristics of those compounds. Polyphenols may interact with proteins both reversibly and irreversibly. Some of the examples from both
interactions in the literature are given in Table 1. In reversible interactions, usually non-covalent forces such as hydrogen bonding, hydrophobic bonding and van der Waals forces are involved (Charlton et al., 2002;
Jobstl, O'Connell, Fairclough, & Williamson, 2004; Poncet-Legrand et al.,
2006; Prigent, Gruppen, Visser, Van Koningsveld, & Alfons, 2003;
Richard, Lefeuvre, Descendit, Quideau, & Monti, 2006; Richard, Vitrac,
Merillon, & Monti, 2005; Siebert, 2006), whereas in irreversible interactions, covalent bonds are formed between the polyphenols and proteins
(Haslam, 1996). A hydrogen bond is the interaction of a hydrogen atom
that is covalently attached to an electronegative atom such as N, O or S
with another electronegative atom, which is primarily an ionic interaction. They are only stable as long as they are protected from water. Van
der Waals interactions are intermolecular interactions affected by the
surrounding solvent. They are dipoleinduced dipole and induceddipoleinduced dipole interactions between neutral atoms in protein
molecules. When two atoms come close to each other, each atom
induces a dipole in the other via polarization of the electron cloud. The
interactions between these induced dipoles have an attractive as well
956
Fig. 2. The structure of various phenolic compounds.
as repulsive component. Depending on the relative number of negatively

and positively charged residues, proteins have either a net negative or a
net positive charge at neutral pH. These charged groups in proteins
are distributed on the surface of the protein molecule. Electrostatic
interactions occur between like charges (repulsive interactions) and
opposite charges (attractive interactions), which are charged groups on
the surface of the protein molecule and the other molecules in the
media. Hydrogen bonding and electrostatic interactions between
various polar groups are not very stable, and their stabilities depend on
maintenance of an apolar environment. Hydrophobic interactions
among nonpolar groups are stronger (Damodaran, 1996). The number
of studies which describe the reversible interactions is more than
the irreversible ones mainly due to the lack of suitable methods for
quantitating the covalent bonds between molecules.
Trombley, Loegel, Danielson, and Hagerman (2011) have suggested
that bioactivities and bioavailability of plant polyphenols and other catechin derivatives may be affected by the covalent interaction between
polyphenols and proteins. Reaction mechanisms and methods for characterizing non-covalent and covalent interactions between polyphenols
and proteins were reviewed thoroughly by Bourvellec and Renard
(2012), very recently.
The interactions of phenolic compounds and proteins are known to
affect the structure of proteins, content of free polyphenols, antioxidant
capacity and bioavailability of phenolic compounds in foods. A better
understanding of phenolic compoundprotein interactions would
help to control the functional properties of proteins in food products
during processing, transportation and storage. In this review, the effects

of proteinphenolic interactions under various conditions on protein
and phenolic compounds' structure and functionality are described.
2. Parameters affecting interactions between protein and
phenolic compounds
There are many parameters that affect proteinphenolic interactions such as temperature, pH, types of proteins, protein concentration,
types and structures of phenolic compounds, salt concentration, and addition of certain reagents. Complex formation of protein and phenolics results from hydrogen binding and hydrophobic interactions (Hagerman
& Klucher, 1986). Specic to proteinphenolic complex, hydrophobic interactions have been considered to be promoted by hydrogen bonding
(Haslam, 1996), as summarized in Table 1.
2.1. Temperature
Temperature can affect hydrogen bondings and causes the formation
of hydrophobic bondings, therefore, it is an important parameter in the
proteinphenolic interactions. Sastry and Rao (1990) observed that temperature had a signicant inuence on the binding of polyphenol-free
11S protein of sunower seed with 5-O-caffeoylquinic acid. The binding
of polyphenol-free 11S protein of sunower seed and 5-O-caffeoylquinic
acid signicantly decreased as the temperature increased from 30 C to
45 C and it completely disappeared at 55 C. Temperature affected
957
Fig. 3. Reactions of a phenolic acid with amino side chains of polypeptides (Strauss & Gibson, 2004).
both the maximum amount of binding points and the binding afnity of
5-O-caffeoylquinic acid to polyphenol-free 11S protein as a result of the
important role of hydrogen bonding in the binding of 5-O-caffeoylquinic
acid by the 11S protein (Sastry & Rao, 1990). Prigent et al. (2003) studied
interactions of bovine serum albumin (BSA) with 5-O-caffeoylquinic acid
at 5, 25 and 60 C. They reported that the binding afnity of 5-Ocaffeoylquinic acid for BSA decreased with temperature. However,
Hoffmann et al. (2006) reported that precipitation of BSA with
procyanidin derivatives was independent of temperature variations.
Tsai and She (2006) evaluated the contribution of phenolprotein
interactions on the antioxidant capacity of peas after immersion
with ve phenolics under different heating conditions between 30
and 70 C. They extracted superoxide dismutase (SOD) enzyme
from peas, formed proteinphenolic interaction complex and measured
SOD activity and binding capacity of this complex with pea protein. SOD
activity found in fresh or processed fruits is very weak as a result of
protein deformation induced by heating time and temperature. They
reported that the heat stability of SOD increased after interacting with
phenolic compounds as a result of the increase in SOD activation energy
caused by the binding of phenolic compounds to protein. It was observed that the binding effect of phenolic compound was increased
with temperature (Tsai & She, 2006).
2.2. pH
The precipitations of complexes resulting from polyphenolprotein
interactions are pH sensitive. The lowest solubility of polyphenol
protein complexes occurred at 0.33.1 pH units below the isoelectric
point of the proteins (Naczk, Grant, Zadernowski, & Barre, 2006). Unlike the temperature, pH affected only the degree of binding but not
the binding afnity for the interaction between polyphenol-free 11S
protein of sunower seed and 5-O-caffeoylquinic acid. Lower pH led
to stronger binding because the dissociation of protein had more
binding sites at lower pH (Sastry & Rao, 1990). The interactions between chlorogenic acid (CGA) and several proteins such as BSA, lysozyme, and -lactalbumin at pH 7 produced non-covalent bonds
and the amount of CGA bound by BSA (per molecule) was somewhat
higher at lower pH (Prigent et al., 2003). With the increase in pH, the
covalent interaction between lysozyme and CGA was stronger due to
the formation of more radicals or quinones from the autoxidation of
CGA at higher pH. The reactive radicals and quinones subsequently
interacted with proteins covalently (Prigent et al., 2003).
Naczk, Oickle, Pink, and Shahidi (1996) studied the effect of pH on
the formation of crude tannin canola extract/BSA, fetuin, gelatine, and
lysozyme complexes. It was suggested that the optimal pH for precipitation varies for different proteins and generally it is close to the isoelectric point of the protein. Rawel, Meidtner, and Kroll (2005) also
observed higher binding afnity for ferulic acid and CGA close to the isoelectric point of BSA. Frazier, Papadopoulou, and Green (2006) failed to
nd an effect of pH on binding of ()-epicatechin to BSA. This was also
supported by results of Papadopoulou, Green, and Frazier (2005) and
Charlton et al. (2002) and suggested that electrostatic interactions are
not a major factor in forming the ()-epicatechin/BSA complex. Indeed,
increased precipitation of protein/polyphenol complexes close to the
isoelectric point may be attributed to the minimum solubility of the
protein at this pH.
958
Table 1
Different types and mechanisms of proteinphenolic interactions.
Type of interaction
Interaction
mechanism
Protein
Phenolic compound
Reversibly
Hydrogen bonding
-lactalbumin
lysozyme
BSA
Hydrogen bonding
Bovine serum
albumin (BSA)
Van der Waals

forces + Hydrogen
bonding
Irreversibly
Non-covalent
forces
Assessment method
References
Procyanidins of medium DP
Procyanidins of
can lead to an undesirable
various degree of
polymerization (DP) decrease of protein solubility,
but may play a positive role
in foam stability
Ferulic acid (FA)
Thermal stability of BSA
increases upon binding with
FA.
Isothermal titration
calorimetry
Prigent et al.,
2009
uorescence, circular
dichroism and isothermal
titration calorimetry
Bovine
-lactoglobulin
()-epigallocatechin
Hydrophobic
binding
-casein in milk
Hydrophobic
binding
Walnut proteins
Hydrogen bonding
and non-polar
hydrophobic
interactions
Sorghum
proteins
Green tea avonoids Number of surface

(catechins)
hydrophobic sites decreased
with phenolics
Walnut phenolics
The presence of phenolic
compounds decreased protein
solubility in walnut our
obtained from whole kernels.
Sorghum tannins
Precipitation of proteins, and
make them insoluble and
indigestible
uorescence spectra, CD
spectra, infrared spectroscopy,
and synchronous
uorescence spectra
Fluorometry analysis,
isothermal titration
calorimetry
SDS-PAGE
Ojha, Mishra,
Hassan, and
Chaudhury
(2012)
Wu et al.
(2011)
Hydrophobic and
hydrophilic
interactions
Milk
-lactoglubulin
Tea polyphenols
The structural stabilization of

protein increased
FTIR, CD, uorescence

spectroscopic methods
Soy protein
Chlorogenic-, caffeic-,
gallic acid, avones,
apigenine,
kaempferol,
quercetin and
myricetin
Circular dichroism, differential

scanning calorimetry (DSC)
Rawel,
Czajka, Rohn,
and Kroll
(2002)
Fish myobrillar
protein
Caffeic acid,
catechin, ferullic
acid and tannic acid
Texture prole analysis, color

measurement, light
transmission, SDS-PAGE
Prodpran et
al. (2012)
Gelatin
Gallic acid and rutin
1) Reduction in lysine,
cysteine and tryptophan, 2)
The isoelectric points shifted
to lower pH, 3) Increase in
molecular weight, 4) More
hydrophilic surface on soy
protein, 5) Inuence on
solubility
1) Enhanced mechanical
properties with phenolic
compounds, 2) Inuence on
properties and appearance of
protein lms
1) Increased gel strength,
thermal stability, 2) Decrease
in swelling
Yan, Li, Zhao,

and Yi (2011)
Non-disulphide
covalent linkages
-lactoglobulin
Sour cherry
phenolics
(antocyanins)
1) Allergenicity of protein
decreased, 2) Digestibility
remained
Cross-linking
Gelatin (Type A)
Phenolic acid,
quercetin, rutin
Milk protein
Caffeic acid
Heat coagulation time-pH

prole, determination of
available lysine, sulfhydryl
groups, zeta potential, relative
viscosity, measurement of
casein micelle size
O'Connell
and Fox
(1999)
Porcine plasma
protein
Tannic acid, caffeic

acid, ferulic acid
1) Mechanical strength of gels

increased, 2) Reduced swelling,
3) Fewer free amino groups,
4) denser polymeric networks
1) Enhance the heat stability of
milk
2) Reduced the lysine and
sulfhydryl content of heated milk
3) No effect on rennet
coagulation time, alcohol
stability, viscosity or
zeta potential
4) Increased casein micelle size
5) Increased crosslinking of milk
proteins
1) Tensile strength increased,
2) Elongation at break
increased, 3) water vapor
permeability of lms increased
Texture prole analysis,

rheometry, DSC, swelling tests,
scanning electron microscopy,
X-ray diffraction, FTIR
SDS-PAGE, isoelectrofocusing,
immunoblotting,
size-exclusion and
reverse-phase chromatography,
mass spectrometry, digestibility,
antioxidant activity
Free amino groups analysis,
gel rigidity, swelling, dynamic
light scattering
Mechanical properties, water

vapor permeability
Nuthong,
Benjakul, and
Prodpran
(2009)
Covalent
bonds
Cross-linking
Changes in functionality or
modication
contribute to the
functionality of the milk
products
Yksel et al.
(2010)
Labuckas et
al. (2008)
Duodu,
Taylor,
Belton, and
Hamaker
(2003)
Kanakis et al.
(2011)
Tantoush et
al. (2011)
Strauss and
Gibson
(2004)
2.3. Types of proteins and protein concentration

Proteinphenolic interaction is affected by the types of proteins and
the molar ratio of phenolic/protein (Prigent et al., 2003). They can bind
either hydrophobically or hydrophilically depending on binding sites of
the protein. The difference in binding afnity among proteins depends
on several factors, such as hydrophobicity (BSA>-lactalbumin >
lysozyme), isoelectric point and the amino acid composition of proteins
(Prigent et al., 2003). For example, the binding afnity of CGA to BSA was
higher than to lysozyme and -lactalbumin. The amount of protein in
the solution affects the proteinphenolic interactions as well. If the concentration of BSA was low, the difference of protein precipitation
between 0.5 mg/ml of BSA and 1.0 mg/ml of BSA was not statistically
signicant. On the other hand, when the concentration of BSA was
higher than 1.0 mg/ml, the protein precipitation effect was signicantly
lower comparing to the effect obtained from higher concentrations of
BSA (Naczk et al., 1996).
959
weakens the afnity for proteins. Glycosylation of resveratrol slightly

reduced the afnity for milk proteins. The afnity of resveratrol for
milk proteins was about 2.51 times higher than that of polydatin. The
hydrogenation of the C2_C3 double bond of avonoids decreased the
binding afnities for milk proteins about 7.24 to 75.86 times. The afnities of apigenin and myricetin for milk proteins were about 75.86 times
and 8.51 times higher than those of naringenin and dihydromyricetin,
respectively. The hydrogenation of the C2_C3 double bond for many
avonoids decreased the binding afnity for BSA by 24 orders of magnitude. Galloylation of catechins signicantly improved the binding afnities with milk proteins about 1001000 times. The pyrogallol-type
catechins showed lower afnities than catechol-type catechins. Moreover, the afnity of catechin with 2,3-trans structure for milk proteins
was found to be higher than that of the catechin with 2,3-cis structure.
The esterication of gallic acid signicantly improved the afnity for milk
proteins. The afnities of gallic acid and its esters with -amylase were
determined as: methyl gallate>ethyl gallate>propyl gallate>gallic
acid (Xiao et al., 2011).
2.4. Types and structures of phenolic compounds

2.5. Other factors
Different types of phenolic compounds affect proteinphenolic interactions depending on factors such as molecular weight, methylation, hydroxylation, glycosylation, and hydrogenation of phenolic compounds.
The binding afnity of polyphenols to proteins increases with their molecular size. Larger polyphenols like those present in black tea (theaavin,
thearubigin) are more likely to bind milk proteins due to the fermentative
oxidation/polymerization of catechin monomers (Dubeau, Samson, &
Tajmir-Riahi, 2010). Among several low molecular weight phenolic compounds including p-coumaric acid, p-hydroxybenzoic acid, cinnamic acids
(protocatechuic acid and caffeic acid) and catechin, the 3,4-dihydroxy
benzoic and cinnamic acids had the strongest binding afnity for BSA
while there was no signicant interaction between p-hydroxybenzoic
acid and BSA (Bartolome, Estrella, & Hernandez, 2000). Although both
quercetin and quercetin 3-O--D-glucopyranoside are avonoids, their
binding afnities with BSA were different since stronger interaction was
observed between BSA and quercetin (Martini, Claudia, & Claudio,
2008). For increasing the heat stability of SOD in peas, hydroxycinnamic
acids including ferulic acid, coumaric acid and caffeic acid were found to
increase the heat stability better than hydroxybenzoic acid; coumaric
acid was found to be superior for enhancing the antioxidant activity of
SOD and showed the strongest binding ability with pea protein (Tsai &
She, 2006).
Xiao et al. (2011) investigated the relationship between the structural properties of dietary polyphenols and their afnities for milk
proteins. Methylation and methoxylation of avonoids weakened or
little affected their binding afnities for milk proteins. In general,
the methylation of hydroxyl group in avonoids decreased their binding afnities for milk proteins by 1.1014.79 times. The afnity of
daidzein for milk proteins was found to be 14.79-times higher than
that of its methylated form (formononetin). 40-methoxylation of
galangin hardly affected the afnity for milk proteins. Hydroxylation
on the rings A and B of avones and avonols slightly enhanced the
binding afnities for milk proteins. The hydroxylation on the ring A
of avanones signicantly improved the afnities for milk proteins.
However, the hydroxylation on the ring C of avones hardly inuenced
the binding afnities for milk proteins and the hydroxylation on ring A
of isoavones reduced or little affected the afnities for milk proteins.
Quercetin binding afnities for milk proteins was found to be only
1.02 times higher than that of quercitrin which is the glycoside formed
from quercetin. The afnities (logKa) of naringenin, naringin and
narirutin for milk proteins were determined as 3.94, 3.76, and 3.64, respectively. It revealed that the monoglycosides of avonoids showed
stronger binding afnities with milk proteins than their polyglycoside
forms. The decreasing afnity for milk protein after glycosylation may
be caused by the non-planar structure. After the hydroxyl group is
substituted by a glycoside, steric hindrance may take place, which
Other factors inuencing the proteinphenolic interactions are

salt concentration and addition of certain reagents. The binding
strength of CGA to sunower 11S protein was reduced with an increase in NaCl concentration. The concentration of NaCl lowered the
amount of binding points instead of affecting the binding afnity
due to fact that salts at high concentration can inhibit the dissociation
of oligomeric proteins (Sastry & Rao, 1990). Some reagents, such as
Na2SO3 (a reducing agent), even at low concentration can affect the
proteinphenolic interactions, the binding between CGA and 11S protein disappeared completely in 0.01 M Na2SO3 (Sastry & Rao, 1990).
3. Effects of proteinphenolic compounds interactions on proteins
3.1. Effects on structure
The mechanism of how proteins inuence polyphenols is still not
yet known. In order to give an explanation, rstly the changes in the
structures of the proteins should be well understood. There are several researches that have been performed recently to understand the
mechanism by which the antioxidants in tea are affected by the addition of milk.
Hasni et al. (2011) studied the interaction of - and -caseins
with tea polyphenols (+)-catechin (C), (+)-epicatechin (EC),
(+)-epigallocatechin (EGC) and (+)-epigallocatechin gallate (EGCG)
at a molecular level, using Fourier transform infrared (FTIR), UVvisible,
circular dichroism (CD), uorescence spectroscopic methods and molecular modeling. It was concluded that tea polyphenols weakly bind
to -casein and -casein through both hydrophilic and hydrophobic interactions. The order of binding increases as the number of OH group increased with C ~ EC > EGC > EGCG. -Casein forms stronger complexes
with tea polyphenols than -casein, due to the more hydrophobic
nature of -casein. Structural modeling of the interaction between and -caseins and tea polyphenols showed that the participation of
several amino acid residues in polyphenolprotein complexation with
extended H-bonding network. Casein conformation was changed by
polyphenol with a major reduction of -helix and -sheet and increase
of random coil (Hasni et al., 2011).
Kanakis et al. (2011) investigated the interaction of -lactogolobulin
(LG) with tea polyphenols (+)-C, ()-EC, ()-ECG and ()-EGCG at
molecular level, using FTIR, CD, uorescence spectroscopic methods
and molecular modeling. They determined polyphenol binding mode,
the binding constant and the effects of polyphenol complexation on
LG stability and secondary structure. As a result of structural analysis
it was observed that polyphenols bind LG through both hydrophilic
and hydrophobic interactions. Tea polyphenols make weak bonds
960
with LG in solution. The order of binding increases as the

number of OH group increased with EGCG> ECG>EC>C. Molecular
modeling showed the participation of several amino acid residues in
polyphenolprotein complexation with extended H-bonding network.
The LG conformation was changed in the presence of polyphenols
with an increase in -sheet and -helix, suggesting stronger structural
stabilization of the protein (Kanakis et al., 2011).
In the study of Wu et al. (2011) binding interaction between EGC
and LG was investigated using uorescence spectra, CD spectra,
infrared spectroscopy, and synchronous uorescence spectra. The
changes in negative entropy and the enthalpy indicated that the
interaction between EGC and LG was driven mainly by van der
Waals interactions and hydrogen bonding. This also indicated that
the surface of LG was covered by EGC, resulting in change of native
conformation of LG (Wu et al., 2011).
Roy et al. (2012) also investigated the interactions of two stereoisomeric antioxidant avonoids, C and EC with BSA and human
serum albumin (HSA) by steady state and time resolved uorescence,
phosphorescence, CD, FTIR and proteinligand docking studies. The
steady-state uorescence studies indicated a single binding site for
both the ligands. FTIR spectra suggest that in both the albumins, C
and EC stabilize the -helix at the cost of a corresponding loss in
the -sheet structure. CD studies have been carried out using (+)-C,
and both the epimers (+)-C and ()-C. The low temperature phosphorescence and proteinligand [(+), () and () forms of C and
EC] docking studies indicated that the ligands bind in the proximity
of Trp 134 of BSA and Trp 214 of HSA, thereby changing their solvent
accessible surface areas (Roy et al., 2012).
It was observed that the proteinphenolic interactions increase
the molecular weight of proteins. In a study of Prigent et al. (2003)
molecular weight of -lactalbumin and lysozyme was observed to
be increased after incubating with CGA at pH 7.0 from 680 to
690 Da. It was suggested that this may be resulted from covalent interactions between proteins and quinones formed by heat oxidation
of phenolic compounds (Prigent et al., 2003). Rawel et al. (2002)
also studied the interactions between the soy proteins and phenolics
such as CGA, caffeic acid and gallic acid. They have found out that
these interactions caused the formation of high molecular weight
fractions as well.
3.2. Effects on functional properties
Proteins have several functional properties in food systems such
as solubility, water absorption and binding, modifying viscosity,
gelation, adhesion, elasticity, plasticity, emulsication, fat absorption,
color and avor binding, foaming, catalysis, and ber formation.
Depending on the food systems and the proteins involved, such
functions may be desirable (such as the use of egg proteins as a
foaming agent) or undesirable (such as enzymatic browning of fruits
and vegetables) for foods. The distinctive functional properties of
various proteins make them crucial for the production of some
foods (e.g., wheat gluten is a unique protein for the elasticity and
plasticity of the dough) (Sathe, 2012).
Protein solubility or insolubility is an important factor for understanding the performance of functionality of the protein in food
systems since protein insolubility may also limit other functional
properties of proteins. Protein solubility depends on some of the
intrinsic (e.g., protein amino acid composition, protein amino acid
sequence) and extrinsic (pH, temperature, ionic strength) factors
(Sathe, 2012). The presence of phenolic compounds also affects protein solubility. Prigent et al. (2003) reported that there was a decrease
in the solubility of lysozyme in the presence of CGA at pH 8.0
according to the oxidation of CGA to form quinones into basic solution. Similar reduction in lysozyme and myoglobin was also observed
after the reaction of proteins and phenolic compounds (Kroll, Rawel,
Rohn, & Czajka, 2001).
Walnut kernels have a signicant amount of phenolic compounds,

which are generally present in the hull. When kernels are wholeground and the oil is extracted, most phenolics remain in the our
where they can precipitate proteins through different mechanisms,
such as hydrophobic and ionic interactions, hydrogen and covalent
bonds. It has been reported that phenolic compounds obtained from
the whole kernels decreased protein solubility. This was affected
strongly by the solvent system. Proteins from whole kernels, especially those extracted with water and NaCl solution, reduced the protein
solubility, suggesting that phenolic compounds bind to proteins when
they are dispersed in aqueous media at neutral pH (Relkin & Shukat,
2012). The study of Van Koningsveld et al. (2002) also conrmed that
phenolic compounds may be responsible for the low solubility of
some potato protein preparations.
The interactions of proanthocyanidins with proteins can also modify the functional properties of food proteins, as these interactions
often result in a decrease of protein solubility. In apple juice, in
which the main phenolic compound is the dimeric procyanidin B2,
it was observed that a higher ionic strength decreases protein solubility after several days of incubation (Tajchakavit, Boye, Blanger, &
Couture, 2001).
In the study of Rawel et al. (2002) soy glycinin and soy trypsin inhibitor were derivatized by chlorogenic and caffeic acid (cinnamic acids,
C6\C3 structure), and by gallic acid representing hydroxybenzoic
acids (C6\C1 structure). Further, the avonoids, avone, apigenin,
kaempferol, quercetin and myricetin (C6\C3\C6 structure) were
also caused to react with soy proteins to estimate the inuence of the
number and the position of hydroxy substituents. The derivatives
were characterized in terms of their solubility at different pH values to
document the inuence on the functional properties.
It was observed that the reaction of phenolic compounds with
proteins may induce cross-linking of the proteins. These interactions
also change the net charge in the protein molecules, which in turn affects the solubility of the derivatives. The secondary and tertiary
structures of the proteins change as a result of these interactions,
inuencing the surface properties of the molecules making them
hydrophilic in nature. This change in hydrophilic/hydrophobic properties may affect not only the solubility behavior, but also other functional properties like emulsication, foaming properties and gelation
of the derivatives (Rawel et al., 2002).
On the other hand, interaction of proteins with phenolic compounds may improve the thermal stability of proteins. Tsai and She
(2006) observed that the thermal stability of SOD was increased
after its interaction with phenolic compounds. Application of higher
temperature resulted in higher binding capacity of phenolic compounds
to proteins. However, heating also led to the disruption of the protein
phenolic complex. It has been reported that SOD activity was higher
after incubating with phenolic compounds, and hydroxycinnamic acids
had more effect than hydroxybenzoic acid on SOD activity; this was due
to the resonance structure of hydroxycinnamic acids having the capability
to support the stability of SOD through incubation. The antioxidant capacity of peas increased after interacting with phenolic compounds and
coumaric acid provided the maximum antioxidant activity to peas
compared to gallic acid, catechin, ferulic acid and caffeic acid (Tsai &
She, 2006). The increase in antioxidant activity in peas was as a result of
proteinphenolic interactions in peas which stabilized the protein, generally SOD, and provided the antioxidant capacity for the protein during
heating (Tsai & She, 2006). It was as a result of the stronger binding of
CGA with the native BSA than the denatured BSA. It was also found
that there was a slight decrease in the denaturation temperature
when lysozyme interacts with CGA. This decrease occurred as a
result of the stronger binding effect between CGA and unfolded lysozyme. Another reason was the enhanced destabilization and
unfolding of lysozyme. Besides, the denaturation temperature and
denaturation enthalpy of -lactalbumin and the denaturation enthalpy of lysozyme were not affected by CGA (Prigent et al., 2003).
A transfer in isoelectric points of soy protein to more acidic pH

values was observed after incubating with different kinds of phenolic compounds (Kroll et al., 2001; Rawel et al., 2002).
In another study, the stabilization of type I collagen was investigated using the plant polyphenol catechin. These results showed a
shrinkage at 70 C implying that catechin was able to impart thermal
stability to collagen (Madhan, Subramanian, Rao, Nair, & Ramasami,
2005).
3.3. Effects on nutritional value and digestibility

A better understanding of the factors that inuence the nutritional
value of proteins is necessary to optimize the biological utilization of
proteins for human and animal nutrition. Nutritional characteristics
of proteins after their interaction with phenolic compounds should
be evaluated.
Polyphenols interact with salivary proteins, especially with prolinerich proteins (PRPs), forming insoluble aggregates that are supposed to
be at the origin of astringency sensation (Soares et al., 2011). Soares,
Mateus, and Fernandes (2012) studied procyanidin trimer and PGG
(pentagalloylglucose) interactions, solubility (both by HPLC) and size
of the complexes formed by dynamic light scattering. The results
showed that mainly acidic PRPs (aPRPs) and statherin interact with
tannins, forming a signicant quantity of complexes (either insoluble
or soluble), while bPRPs (basic PRPs) interact poorly with procyanidin
trimer and gPRPs (glycosylated PRPs) only complex with PGG. In general, PGG formed a high amount of insoluble complex with salivary proteins while procyanidin trimer formed soluble complexes (except for
statherin). These results highlighted the inuence and mechanisms of
different tannins and salivary proteins that could present in their interaction, and consequently in the development of the astringency sensation (Soares et al., 2012).
Rawel et al. (2002) reported reductions in the amounts of lysine,
cysteine and tryptophan determined in soy proteins after interacting
with different phenolic compounds. Soy glycinin and soy trypsin inhibitor were derivatized by chlorogenic and caffeic acids (cinnamic acids,
C6\C3 structure), and by gallic acid representing hydroxybenzoic
acids (C6\C1 structure). Further, the avonoids, avone, apigenin,
kaempferol, quercetin and myricetin (C6\C3\C6 structure) were
also reacted with soy proteins. They have estimated the inuence of
the number and the position of hydroxy substituents of these avonoids
on selected physicochemical properties of the soy proteins. The derivation caused a reduction of lysine, cysteine and tryptophan residues
in soy proteins. These results underlined the possible nutritional consequence of proteinphenolic interactions by affecting the bioavailability
of the essential amino acids in the food systems (Rawel et al., 2002).
It has been also reported that the presence of condensed tannins
decreased in vivo and in vitro digestibility of proteins. Their interactions form indigestible proteins and inhibitory digestive enzymes. It
was observed that sorghum-condensed tannins can complex karin
which is one of the main proteins of sorghum. As a result of this complexation, a decrease in protein digestibility of high-tannin sorghum
was observed (Emmambux & Taylor, 2003). Duodu et al. (2003)
also reviewed the factors affecting the sorghum digestibility and classied the phenolic compounds depending on the exogenous factors
affecting protein digestibility.
In another study, leaves harvested from Acacia drepanolobium,
Acacia nilotica, Acacia seyal, Acacia tortilis, Acacia polyacantha and
Acacia senegal were studied and it was observed that tannins have
negative inuence on digestibility of proteins in vitro (Rubanza et
al., 2005). Similarly, another study, on the effect of sea buckthorn
procyanidins on the protein digestibility also demonstrated that
sea buckthorn procyanidins precipitated proteins and inhibited digestive enzymes which may have changed the digestion of proteins
(Arimboor & Arumughan, 2011).
961
4. Effects of proteinphenolic compounds interactions on

phenolic compounds
The antimicrobial activity of polyphenols has been extensively investigated against a wide rande of microorganism. Among polyphenols, avan-3-ols, avonols and tannins received most attention due
to their wide spectrum and higher antimicrobial activity in comparison with other polyphenols. They are able to suppress a number
of microbial virulence factors (such as inhibition of biolm formation,
reduction of host ligands adhesion, and neutralization of bacterial
toxins) and show synergism with antibiotics (Daglia, 2012). Proteinpolyphenol interactions may decrease the antimicrobial capacities of
polyphenols. Von Staszewski, Pilosof, and Jagus (2011) evaluated the
changes of antimicrobial capacities of different Argentinean green tea
varieties by addition of whey proteins. The results revealed some degree of masking in the antimicrobial activity of green tea infusions
when whey proteins are added. The antimicrobial effects in the presence of whey proteins correlated with the polyphenol content of the
green tea infusions and increased with the reduction of whey protein
concentration. The antimicrobial effect and potential was similar within
a pH range from 4.0 to 7.0, allowing its application to a wide group of
foods (Von Staszewski et al., 2011). There are several other studies dealing with the changes that occur as a result of proteinphenolic interactions. However, in this review mainly effects on antioxidants and
bioavailability of phenolic compounds are evaluated.
4.1. Effects on total phenolic content (TPC), total avonoid content (TFC)
and antioxidant activity
The measurement of the antioxidant capacity of food products is a
matter of growing interest because it may provide a variety of information, such as resistance to oxidation, quantitative contribution of
antioxidant substances, or the antioxidant activity that they may
present inside the organism when ingested (Huang, Ou, & Prior,
2005; Serrano, Goi, & Saura-Calixto, 2007). Numerous in vitro studies have been conducted to evaluate the total antioxidant capacity
(TAC) of food products. So far, however, there is no ofcial standardized method, and therefore it is recommended that each evaluation
should be performed under different oxidation conditions and different measurement methods. The methods for measuring antioxidant
capacity are basically classied into two groups, depending on the reaction mechanism: methods based on hydrogen atom transfer and
methods based on electron transfer (Huang et al., 2005). The majority
of hydrogen atom transfer-based assays apply a competitive scheme,
in which antioxidant and substrate compete for thermally generated
peroxyl radicals through the decomposition of azo-compounds. Electron transfer based assays measure the capacity of an antioxidant in
the reduction of an oxidant, which results in color changes when oxidation occurs. The degree of color change is correlated with the
sample's antioxidant concentration (Zulueta, Esteve, & Frigola, 2009).
In recent years, a wide range of spectrophotometric assays has
been adopted to measure antioxidant capacity of foods, the most popular ones are 2,2-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid
(ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, among
others such as oxygen radical absorbance capacity (ORAC), copper reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays. These antioxidant capacity measurement
assays are used widely for all kinds of foods such as vegetables and
fruits, cocoa and cocoa products, beverages including fruit juices, tea
and coffee etc. There are many studies on the effect of proteins on
the antioxidant capacity of phenolic compounds inuenced by the
proteinphenolic compound interactions. A brief outline on the effects
of proteinphenolic compound interactions on the total phenolic and
avonoid contents and total antioxidant capacities of polyphenol rich
food products including the methods of analysis are given in Table 2.
962
Almajano et al. (2007) mixed BSA, -lactoglobulin, -lactalbumin,

-casein and -casein with epigallocatechin gallate (EGCG) at 30 C,
resulted with the formation of an adduct having antioxidant activity.
The antioxidant activity of the protein component, which included
both unmodied protein and the proteinEGCG adduct, increased
with storage time at 30 C using ABTS, FRAP and ORAC methods
(Almajano et al., 2007).
Belak et al. (2009) studied the total phenolic contents, total avonoid contents and antioxidant capacities of various chocolate products.
They reported that the lowest total phenolic content, total avonoid
content and antioxidant capacities were observed in milk chocolate
although it contains higher cocoa solids content (29%) than cocoa bars
(16%). It was suggested that this decrease was as a result of strong
catechinprotein interactions. Milk based products represent a very
complex matrix where strong catechinprotein interactions are wellknown to occur and it directly inuences catechin determination by signicantly reducing analytical recovery from the food matrix (Belak
et al., 2009).
Table 2
Effect of proteinphenolic interactions on total phenolics, avonoids and antioxidant capacity.
Results
Reference
(+) TAC
(+) TAC
(+) TAC
Almajano et al.
(2007)
() TP
Milk chocolate (29% cs)
b cocoa bar (16%cs)
() TF
b cocoa bar (16%cs)
() TAC
b cocoa bar (16%cs)
() TAC
b cocoa bar (16%cs)
() TAC
b cocoa bar (16%cs)
() TP
() TAC (6.0%, 8.3%, and 19.6%)
() TAC 4 times larger results
than ABTS
(+) TAC 19%, 10% and 12%
Belak, Komes,
Hori, Gani, and
Karlovi (2009)
() TP
() TAC
Black tea > black
tea + sugar >black
tea + milk > black
tea + milk + sugar
-carotene-linoleic-acid assay
Sharma, Vijaykumar,
and Jaganmohanrao
(2008)
() TAC
Arts et al. (2002)
DPPH
Whole kernel b hull

Whole kernel b hull
Labuckas et al.
(2008)
Tea phenolics
FRAP
() TAC
Ryan and Petit

(2010)
Black tea phenolics
FRAP
Ryan and Sutherland

() TAC (semi-skimmed
(2011)
bovine milk + tea)
0/(+) TAC (soya milk + black tea)
() TP
Von Staszewski et al.
(2011)
() TP
Niseteo, Komes,
() TF
Belak-Cvitanovi,
() TAC
Hori, and Bude
() TAC
(2012)
() TP
Sanchez-Gonzalez,
() TAC
Jimenez-Escrig, and
() TAC
Saura-Calixto (2005)
() TAC
Ziyatdinova,
Nizamova, and
Budnikov (2010)
Product
Proteins
Phenolics
Antioxidant
capacity
measurement
methods
Milk proteins and

BSA & EGCG
Bovine serum
albumin (BSA)
-lactalbumin,
-lactoglobulin,
-casein,
-casein.
Milk proteins
Epigallocatechin gallate (EGCG)
ABTS
FRAP
ORAC
Dairy products &

confectionary
products
Total
phenolic
content
assays
TP
Cocoa liquor, cocoa powder,

chocolate with (88, 72, 60%) cocoa
solids (cs), cooking chocolate,
powdered chocolate, milk chocolate
(29% cs), cocoa bar (16% cs)
TF
ABTS
DPPH
FRAP
Dairy products & tea Milk proteins

(2% skim milk)
Tea polyphenols (Green tea,

Darjeeling tea, English breakfast
tea respectively)
TP
ABTS
Voltammetry
Lipid
peroxidation
Tea polyphenols
TP
DPPH
(sugar + milk)
(+) TAC
Walnut kernels
Walnut proteins
(mostly glutelin
and globulin)
Dairy products & tea Milk
(semi-skimmed
and skimmed
milk)
Dairy products/soya Semi-skimmed
milk & tea
bovine milk
Soya milk
Whey protein & tea Whey protein
(8% or 16% w/v)
Coffee & milk
Milk proteins
Tea polyphenols (Green and

black tea)
Walnut phenolics
ABTS
TP
Argentinean black tea
TP
Espresso, Turkish, Instant

and Filter Coffee
TP
TF
ABTS
FRAP
Coffee & milk
Coffee & tea & milk
Milk proteins
Milk proteins
Espresso
Tea and coffee
TP
ABTS
FRAP
Coloumetric
FRAP method
(): decrease, (+): increase, TAC: total antioxidant capacity, TP: total phenolics, TF: total avonoids.
Dubeau et al. (2010)
Studies investigating the effect of milk on antioxidant capacities of

tea samples have presented contradictory results, possibly because of
the different methods used for the measurement of antioxidant capacity. Dubeau et al. (2010) measured the antioxidant capacities by
using three complementary assays; ABTS free radical scavenging,
voltammetry, and lipid peroxidation inhibition. They observed that
according to ABTS and voltammetry methods, milk decreased the antioxidant capacities of teas. In contrast, in the lipid peroxidation method,
the results indicated that milk enhanced the chain-breaking antioxidant
capacity of teas. The researchers explained these ndings as milk can
have dual effects on the antioxidant capacity of tea; an inhibitory effect
for reactions taking place in solution or at a solidliquid interface and an
enhancing effect for those in oil-in-water emulsions (Dubeau et al.,
2010).
A similar interaction between the tea phenolic compounds and milk
proteins has also been reported by Sharma et al. (2008) for black tea,
with sugar, milk or both added. They reported that black tea extracts
had the maximum amount of polyphenol content followed by black
tea with sugar and black tea with both milk and sugar. In addition,
total polyphenol contents were found to give the lowest values in
black tea with milk compared to other samples, probably due to either
covalent or non-covalent interactions between plant phenolics and proteins. Arts et al. (2002) also reported that pure , , and -caseins
masked the ABTS-scavenging capacities of green and black tea extracts
and of some pure avonoids typically found in teas to different extents
probably because of covalent or non-covalent interactions. Both interactions might lead to precipitation of proteins through multisite interactions and multidentate interactions. In multisite interactions, several
phenolics bind to one protein molecule and in multidentate interactions
one phenolic binds to several protein sites or protein molecules. These
interactions may cause masking of polyphenol contents on the proteins
(Arts et al., 2002).
Labuckas et al. (2008) studied the interactions of phenolics of walnut kernel proteins (mostly glutelins and globulins) and their effect
on total phenolic content and total antioxidant capacity using the
DPPH (2,2-diphenyl-1-picrylhydrazyl) method. They reported that
the whole kernel has a lower antioxidant capacity and total phenolic
content compared to the hull. This is because the hull contains most
of the phenolics and, therefore, their interactions with proteins may
have decreased the total antioxidant capacity in the whole kernel
(Labuckas et al., 2008).
Ryan and Petit (2010) reported that the additions of milk to tea
signicantly decreased the total antioxidant capacity depending on
the amount of milk compared to the additions of same volume of
water as analyzed by the FRAP method. Besides, antioxidant activity
was decreased with increased amount of added milk. On the other
hand, Ryan and Sutherland (2011) studied the effect of adding soy
milk or bovine milk on the total antioxidant capacity of ve brands of
tea. It was observed that when compared to tea with semi-skimmed
bovine milk, each of the ve brands of analyzed tea samples had significantly higher total antioxidant capacities or no change after the addition of soy milk. It was suggested that the addition of soy milk to
black tea might be a useful alternative to semi-skimmed bovine milk
if maintaining the overall antioxidant potential of the tea infusion is desired (Ryan & Sutherland, 2011).
Von Staszewski et al. (2011) studied the total phenolic contents,
antioxidant and antimicrobial activities of different Argentinean
green tea varieties and whey protein mixtures. Their results revealed
that when whey proteins are mixed with tea infusions, antioxidant
and antimicrobial activity of green tea infusions were masked. The
degree of inhibition of antioxidant activity in each variety did not depend on the total polyphenol concentration, indicating the importance of the particular polyphenol composition of each variety (Von
Staszewski et al., 2011).
Niseteo et al. (2012) studied the total phenolic and total avonoid
contents and antioxidant capacity of different coffee brews and the
963
effects of milk addition. The results revealed that in comparison to

plain brews prepared only with water, the addition of milk decreased
the TPC and TFC of coffee and decaffeinated coffee brews, as opposed
to instant cappuccino brews prepared with milk, which exhibit higher
TPC when compared to plain water-made brews. Milk containing food
products represent a very complex matrix where strong polyphenol
protein interactions are well known to occur (Siebert, Troukhanover,
& Lynn, 1996) and can directly interfere with accurate polyphenol determination by signicantly reducing analytical recovery, which may
be the reason for the overestimation of milk-prepared cappuccino
brews. Contrary to the results obtained for coffee brews prepared
with milk, instant cappuccino brews made with milk exhibited higher
TPC and TFC than those made with water. The higher values of TPC
and TFC in cappuccino brews with milk can be explained by the presence of milk derived compounds and reduced analytical accuracy, as
well as the lack of selectivity of the FolinCiocalteu reagent used for
TPC analysis which reacts not only with phenols but also with other reducing compounds such as carotenoids, amino acids, sugars and vitamin C (Vinson, Su, Zubik, & Bose, 2001). Apart from instant coffee, the
composition of instant cappuccino includes milk powder, powdered
whey proteins, lactose, sugar, vegetable fat, salt, aroma and E99 (calcium lactobionate), ingredients that can interfere with the determination
of phenolic compounds in this assay, thus contributing to the inconsistency of the results. The antioxidant capacities of coffee brews obtained
by both ABTS and FRAP assays were in accordance with TPC and TFC
values and as expected, decreased with the addition of milk. This can
be explained by the fact that up to 1/3 of the quantity of CGA, as the
main antioxidant compound, interacts with milk proteins in coffee
(Dupas, Marsset-Baglieri, Ordonaud, Ducept, & Maillard, 2006).
Another study on coffee brews (Sanchez-Gonzalez et al., 2005) was
also in agreement with the results of Niseteo et al. (2012) The researchers studied the effect of milk on the total phenolic content and antioxidant capacity of espresso samples by ABTS and DPPH assays. They
observed that when milk was added to the coffee, antioxidant activity
decreased depending on increasing amount of milk (Sanchez-Gonzalez
et al., 2005).
Ziyatdinova et al. (2010) developed a new approach for the evaluation of total free polyphenols content in the presence of proteins
using electrogenerated [Fe(CN)6] 3 ions as coulometric titrant. Mixtures of tea and coffee were prepared for further evaluating the effect
of milk proteins on the antioxidant properties of beverages. Masking
effect of milk proteins on ferric reducing power of tea and coffee
reecting the content of free polyphenols in the system was observed.
Similar masking effect of milk was also observed for the instant coffee
(Ziyatdinova et al., 2010).
4.2. Effects on the content of individual phenolic compounds
There are very few studies found in the literature about the effects
of proteinphenolic interactions on the content of individual phenolic
compounds. These studies should be encouraged to understand the
exact mechanism behind the proteinphenolic interactions.
Ferruzzi and Green (2006) studied the interactions of tea catechins with proteins. Their objective was to evaluate the effect of pepsin treatment as an enzymatic step to increase catechin recovery from
milk and other formulations rich in protein. They combined brewed
green tea with skimmed milk in ratios of 10% to 50% and analyzed
tea catechins by reversed phase High Performance Liquid Chromatography (HPLC) with photodiode array detection (PDA). The results indicated that recovery decreased with increasing milk content. The
presence and ratio of milk most affected gallated catechins, specically epigallocatechin-gallate (EGCG) and epicatechin-gallate (ECG).
Bartolome et al. (2000) prepared mixtures of BSA and several low
molecular weight phenolics that were incubated and fractionated
using G-50 Sephadex chromatography. Among the selected commercial phenolic standards tested (p-coumaric acid, p-hydroxybenzoic
964
acid, protocatechuic acid, caffeic acid and (+)-catechin), the strongest

BSA-binding afnity was demonstrated by 3,4-dihydroxy benzoic and
cinnamic acids (protocatechuic acid and caffeic acid), whereas
p-hydroxybenzoic acid did not interact with BSA. Moreover, the same
methodology was also applied to a phenolic extract obtained from lentils containing p-coumaric acid, a p-coumaric acid derivative, (+)-catechin and procyanidins B3 and B1. Interactions between lentil phenolic
extracts and BSA were comparable to those observed among the commercial standards and BSA (Bartolome et al., 2000).
According to the results of these studies, it is obvious that different
phenolic compounds might show different behaviors during their interactions with proteins. Taking into account that there are many
types of phenolic compounds present in food systems, further studies
on individual phenolic compounds are of critical importance to understand their exact role and behavior.
4.3. Effects on in-vivo bioavailability
Proteins affect the antioxidant activity of polyphenols mostly in a
negative way. They usually decrease the antioxidant activity due to
their strong binding afnity to polyphenols. However, the effect of
proteinpolyphenolic compound interactions should also be considered from the bioavailability of phenolic compounds point of view.
It is important to determine whether protein interaction with polyphenols modulate the uptake and concentration of polyphenols in
plasma. A brief outline on the effects of proteinphenolic interactions
on the bioavailability of phenolics is presented in Table 3.
Keogh et al. (2007) conducted experimental studies with human
subjects consisting of 24 middle-aged men and women evaluating
whether milk proteins with cocoa polyphenols modulate the uptake
and concentration of polyphenols in plasma. It was proved that milk
powder did not inuence the average concentration of polyphenols.
Although it slightly accelerated absorption, this was of no physiological signicance (Keogh et al., 2007).
Duarte and Farah (2011) also studied the effect of simultaneous consumption of coffee and milk on the urinary excretion of CGA and metabolites. Experimental subjects were submitted to consumption of water,
instant coffee dissolved in water and instant coffee dissolved in milk.
After 24 h of consumption, the urine of the subjects was collected to analyze CGA and metabolites by HPLC/LCMS (liquid chromatography-mass
spectrometry). It was observed that the amount of CGA and metabolites
recovered after consumption of combined coffee-milk (40%27%) was
consistently lower in all subjects compared to that of coffee alone
(68%20%). It was suggested that the simultaneous consumption of
milk and coffee may impair the bioavailability of coffee CGA in humans
(Duarte & Farah, 2011).
In another study conducted in vivo, it was investigated whether the
interaction of milk reduces the bioaccessibility of tea catechins associated with tea benecial effects. Addition of milk to black tea resulted with
the formation of polyphenolprotein complexes, and a decrease in total
catechin recovery. Polyphenolprotein complexes were degraded during digestion. It was very unlikely that consumption of tea with or without milk will result in a difference in catechin plasma concentration.
This result is in disagreement with other studies in which a decrease
in the polyphenol bioavailability was observed by the effect of
proteinphenolic interactions (Burg-Koorevaar et al., 2011).
Kyle et al. (2007) also studied the addition of milk to tea on absorption of polyphenols. It was found that consumption of black tea
was associated with signicant increases in plasma antioxidant capacity
and concentrations of total phenols, catechins, and the avonols quercetin and kaempferol within 80 min. However, the polyphenol absorption was unaffected by the addition of milk to black tea (Kyle et al.,
2007).
Serani et al. (2009) studied the bioavailability of phenolics and in
vivo antioxidant capacity of blueberries consumed with and without
milk by conducting the experiments with eleven healthy human
volunteers who consumed either 200 g of blueberries plus 200 ml

of water or 200 g of blueberries plus 200 ml of whole milk. The results showed that ingestion of blueberries increased plasma levels
of reducing and chain-breaking potential and enhanced plasma concentrations of caffeic and ferulic acids. However, there was no increase in plasma antioxidant capacity when blueberries and milk
were ingested. There was a reduction in the peak plasma concentrations of caffeic and ferulic acid as well as the overall absorption of
caffeic acid. As a conclusion of this study, the researchers suggested
that milk and blueberry interactions impair the in vivo antioxidant
properties of blueberries and reduce the caffeic acid absorption
(Serani et al., 2009).
On the other hand, there are contradictory results about the effect of
milk on the plasma antioxidant capacity as reviewed by Lotito and Frei
(2006). It has been suggested that polypeptide chains are located
around the fat globule membrane, which promotes their linkage with
phenolic compounds (Serani et al., 2009) and probably results with a
decrease in the accessibility of the colonic microbiota and thus their
degradation (Lotito & Frei, 2006). Recently, the same behavior was observed in yogurt. Roowi et al. (2009) investigated the catabolism of
hesperetin-7-O-rutinoside following acute supplementation of orange
juice, with and without yogurt. The orange juice was found to contain
hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside. GCMS
(gas chromatographymass spectrometry) analysis of the urine samples resulted with the identication of nine phenolic acids, ve of
which, 3-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid,
dihydroferulic acid, 3-methoxy-4-hydroxyphenylhydracrylic acid and
3-hydroxyhippuric acid, were related with orange juice consumption signifying that they were derived from colonic catabolism of hesperetin7-O-rutinoside. The overall (024 h) excretion of the ve phenolic acids
increased signicantly, equivalent to 37% of the ingested avanones, following orange juice consumption. When the orange juice was ingested
with yogurt, excretion fell back markedly (Roowi et al., 2009).
In another study, Green et al. (2007) characterized the effect of common food additives on digestive recovery of tea catechins. Green tea
water extracts were prepared containing EC, EGC, EGCG and ECG, respectively. Bovine, soy rice milks were added to understand the impact
of creaming agents on catechin digestive recovery. Samples were analyzed with in vitro digestive method which simulated gastric and
small intestinal conditions with pre- and post- digestion catechin proles measured by HPLC. Tea samples enriched with 50% bovine, soy
and rice milk increased the recovery of total catechins by 52, 55 and
69%, respectively. On the other hand, in vivo methods which represent
the dynamic nature and heterogeneity of the gastrointestinal tract did
not give any signicant difference between the control sample and
teas enriched with bovine, rice and soy milk. According to the results,
there were no signicant differences found in catechin bioavailability
and plasma antioxidant activity between tea and tea-milk beverage in
humans in in vivo observations. As a result, it was concluded that the results of in vitro methods cannot be fully and directly extended to in vivo
effects (Green et al., 2007).
Arts et al. (2001) studied the interactions between human blood
plasma and quercetin, rutin, catechin and 7-monohydroxyethylrutoside
using the ABTS method. They found that the antioxidant capacity of
human blood plasma enriched with these phenolic compounds was
lower than the sum of antioxidant capacities of both components. This
effect was found to be much lower in deproteinated plasma. Therefore,
it was attributed to the interactions of catechols with human blood
plasma.
Leenen et al. (2000) also studied the effect of milk addition on the
plasma antioxidant capacity of black and green tea samples in vivo.
Blood samples were obtained at baseline and at several time points
up to 2 h post-tea drinking. Plasma was analyzed for total catechins
and antioxidant activity, using the FRAP assay. The results indicated
that addition of milk to black or green tea did not signicantly affect
the plasma antioxidant activity. In another study by Reddy et al.
965

Table 3
Effects of proteinphenolic interactions on bioavailability of phenolics.
Product
Proteins
Phenolics
Methods
Results
Reference
In vivo methods
Dairy products
& cocoa
Milk proteins
Cocoa phenolics (catechins

and epicatechins)
(=) for C and EC

(=) B
Keogh, McInerney,
and Clifton (2007)
Milk proteins
Coffee phenolics (CGA and

metabolites)
Tea phenolics (English and
Indian tea catechins)
HPLC (Blood samples at 0, 0.5, 1, 1.5,

2.3, 4, 6, 8 h, catechin (C) and
epicatechin(EC) analysis)
HPLC/LCMS (urine samples after
24 h)
gastric, small intestinal, and brush
border digestion, total catechin
(TCAT) recovery
HPLC (blood samples 10 min before
the volunteer drank one of the test
beverages, then 50, 80, and 180 min
thereafter)
FRAP
Reversed phase (RP)-HPLC
()
()
()
()
CGA and metabolites

B
TCAT
B
(=)
(=)
(=)
(=)
TCAT
quercetin
kaempferol
B
Duarte and Farah

(2011)
Burg-Koorevaar,
Miret, and
Duchateau (2011)
Kyle, Morrice,
McNeill, and
Duthie (2007)
Dairy products
& coffee
Dairy products
& tea
Dairy products
& tea
Milk proteins
(skimmed or
full-fat milk)
Milk proteins
Tea phenolics (catechins,

quercetin, kaempferol)
Fruits & tea
Milk proteins
Blueberry fruit phenolics
Dairy products
& fruit juices
Yogurt proteins
Orange juice phenolics

(hesperetin-7-O-rutinoside
and
naringenin-7-O-rutinoside)
GC-MS analysis of urine (24 h)
Dairy products
& tea
Bovine, rice and

soy milk
proteins (50%)
HPLC
pre- and post-digestion catechin
proles
in vivo methods
Blood plasma
& antioxidants
Arts et al. (2001)
Blood plasma
& antioxidants
Human blood
plasma proteins
Green tea phenolics

epicatechin (EC),
epigallocatechin (EGC),
epigallocatechin-gallate
(EGCG) and
epicatechin-gallate (ECG)
quercetin, rutin, (+)
catechin,
Dairy products
& coffee
Dairy products
& tea
Milk proteins
Milk proteins
Black and green tea

phenolics
Dairy products
& tea
Milk proteins
Dairy products
& coffee
() TAC
() caffeic acid
() ferulic acid
() B
() B of 5 phenolic acids
(3-hydroxyphenylacetic acid,
3-hydroxyphenylhydracrylic acid,
dihydroferulic acid,
3-methoxy-4-hydroxyphenylhydracrylic
acid and 3-hydroxyhippuric acid)
() TCAT, () B
(=) TCAT, (=) B
Serani et al. (2009)
Roowi, Mullen,
Edwards, and
Crozier (2009)
Green, Murphy,
Schulz, Watkins,
and Ferruzzi (2007)
7-monohydroxyethylrutoside
In vivo methods, ABTS
() TAC, () B
In vivo methods, ABTS
() TAC, () B
Bauz et al. (2008)
HPLC (plasma samples)

In vivo methods
In vivo methods, FRAP
(=) CGA, (=) B

(=) CQA, (=) B
(=) plasma antioxidant activity
Dupas et al. (2006)
Black tea phenolics
In vivo methods
(=) plasma antioxidant activity
Whole milk
or nondairy
creamer
Instant coffee phenolics
Dairy products
& tea
Milk proteins
(=) caffeic acid (CA), ferulic acid (FA),

and isoferulic acid (iFA) equivalents
(=) CGA
(=) B
(=) plasma phenolics
(=) plasma phenolics
(=) B
Dairy products
& tea
Milk proteins
Black and green tea

phenolics (quercetin and
kaempferol glycosides)
Black tea phenolics
Confectionary
& milk
Dairy products
& cocoa
Milk proteins
Milk proteins
Chocolate phenolics
(avan-3-ols)
Cocoa powder avonoids
In vivo methods
Liquid chromatographyElectrospray ionization-tandem
MS analyses and quantication
of coffee phenolics in plasma
HPLC (human plasma samples
every 2 h)
In vivo methods
Flow-mediated dilation (FMD)
nitro-mediated dilation (NMD)
measured by high-resolution
vascular ultrasound, also
performed in isolated
rat aortic rings and endothelial
cells (before and 2 h after
consumption)
HPLC
Human in vivo pharmacokinetics
RP-HPLC
LCMS/MS (urine samples (06,
612, and 1224 h)).
Dairy products
& cocoa
Dairy products
& chocolate
Milk proteins
Cocoa powder avonoids
Milk proteins
Chocolate avonoids
Human blood
plasma proteins
Antioxidants (albumin,
ascorbic acid, GSH, GSSG,
melatonin, plasma,
quercetin, uric acid)
Coffee phenolics (CGA)
LCMS/MS (plasma samples after

2 h of intake)
In vivo methods
FRAP
() FMD
() favorable health effects of tea
on vascular function
() B
Leenen, Roodenburg,
Tijburg, and Wiseman
(2000)
Reddy, Sagar,
Sreeramulu, Venu,
and Raghunath
(2005)
Renouf et al. (2010)
Hollman, Van Het

Hof, Tijburg, and
Katan (2001)
Lorenz et al. (2007)
(=) B (avan-3-ols)
Neilson et al. (2009)
Phenolic acids
() 3,4-dihydroxyphenylacetic,
protocatechuic, 4-hydroxybenzoic,
4-hydroxyhippuric, hippuric, caffeic,
and ferulic acids
(+) vanillic and phenylacetic acids
(=) ()-EC
Urpi-Sarda et al.
(2010)
()()-EC
Serani et al. (2003)
Roura et al. (2007)
(continued on next page)
966
Table 3 (continued)
Product
Proteins
Phenolics
Methods
Results
Reference
In vivo methods
Dairy products
& cocoa
Milk proteins
Cocoa beverage avonoids
In vivo methods
HPLC
(=) ()-EC
Milk proteins
Cocoa avonols
In vivo methods
HPLC
(=) avonols
Schroeter, Holt,
Orozco, Schmitz,
and Keen (2003)
Schramm et al.
(2003)
Milk proteins
Coffee phenolics (CGA)
Dupas et al. (2006)
Bovine serum
albumin
Sea buckthorn
proanthocyanidins
Caco-2 cell model coupled with an in (=) CGA, (=) B

vitro digestion process
(=) CQA, (=) B
In vitro digestion
() B
In vitro Digestion
Caco-2-cell culture experiments
Colourimetric method by using
electrogenated bromine
(=) B (avan-3-ols)
Neilson et al. (2009)
() B
Nizamova, Ziyatdinova,
and Budnikov (2011)
Dairy products
& cocoa
In vitro methods
Dairy products
& coffee
Fruits & bovine
serum
albumin
Confectionary
& milk
Dairy products
& tea
Milk proteins
Chocolate phenolics
(avan-3-ols)
Milk proteins
Black and green tea
(casein and BSA) phenolics (quercetin,
rutin)
Arimboor and
Arumughan (2011)
(=): no signicant difference, (+): increase, (): decrease, B: bioavailability of phenolics, TCAT: total catechins, TAC: total antioxidant capacity.
(2005), the ndings were in agreement with the results of Leenen et

al. (2000). They investigated the effect of milk addition to black tea on
the ability to modulate oxidative stress and antioxidant status in
adult male human volunteers. It did not affect the benecial effects
of black tea on total plasma antioxidant activity, and plasma resistance to oxidation induced ex vivo. It decreased plasma and urinary
thiobarbituric acid reactive substance levels. The results suggested
that the addition of milk may not obviate the ability of black tea to
modulate the antioxidant status of subjects and that consumption of
black tea with/without milk prevents oxidative damage in vivo
(Reddy et al., 2005).
In another study, eighteen healthy volunteers consumed two out
of four supplements for three days: black tea, black tea with milk,
green tea, and water. A cup of the supplement was consumed every
2 h each day for a total of 8 cups per day. The supplements provided
about 100 mol quercetin glycosides and about 6070 micromol
kaempferol glycosides. The addition of milk to black tea did not affect
the plasma concentration of quercetin or kaempferol. As a result, it
was concluded that avonols were absorbed from tea but their bioavailability was not affected by the addition of milk (Hollman et al.,
2001).
Renouf et al. (2010) also studied the effect of adding whole milk or
sugar and creamer to coffee on the bioavailability of coffee phenolics.
According to the results, in comparison to regular black instant coffee,
the addition of milk did not signicantly alter the maximum plasma
concentration (Cmax), or the time necessary to reach Cmax (tmax).
The Cmax of caffeic acid and isoferulic acid was signicantly lower
and the tmax of ferulic acid and isoferulic acid was signicantly longer
for the sugar/nondairy creamer group than for the instant coffee
group. In conclusion, adding whole milk did not change the overall
bioavailability of coffee phenolic acids, whereas sugar and nondairy
creamer affected the tmax and Cmax values but not the appearance of
coffee phenolics in the plasma (Renouf et al., 2010).
Neilson et al. (2009) studied the effect of milk addition to chocolate on the bioavailability of EC of chocolate. EC bioavailability was
assessed from chocolate confections [reference dark chocolate
(CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa beverages [sucrose milk protein (BSMP), non-nutritive sweetener milk
protein (BNMP)], in humans. These results suggested that the presence of milk solids did not signicantly change the bioavailability of
EC among chocolate confection products (Neilson et al., 2009).
Urpi-Sarda et al. (2010) studied the effect of milk on the bioavailability
of cocoa avonoids considering phase II metabolites of epicatechin. They
studied the effect of milk at the colonic microbial metabolism level of
the non-absorbed avanol fraction that reaches the colon and is metabolized by the colonic microbiota into various phenolic acids. Phenolic acids
were analyzed by LCMS/MS after solid-phase extraction. Of the 15 metabolites assessed, the excretion of 9 phenolic acids was affected by the intake of milk. The urinary concentration of 3,4-dihydroxyphenylacetic,
protocatechuic, 4-hydroxybenzoic, 4-hydroxyhippuric, hippuric, caffeic,
and ferulic acids diminished after the intake of cocoa with milk, whereas
urinary concentrations of vanillic and phenylacetic acids increased. In
conclusion, milk moderately affected the formation of microbial phenolic
acids derived from the colonic degradation of procyanidins and other
compounds present in cocoa powder (Urpi-Sarda et al., 2010).
Roura et al. (2007) studied the effect of milk on the absorption of
()-epicatechin (()-EC) from cocoa powder in healthy humans.
Quantication of ()-EC in plasma was determined by LCMS/MS
analysis after a solid-phase extraction procedure. Milk solution, as
one of the most common ways of cocoa powder consumption,
seems to have a negative effect on the absorption of polyphenols
but it was not statistically signicant (Roura et al., 2007).
There are many other studies on the bioavailability of polyphenols
from cocoa that have yielded contradictory results. Serani et al.
(2003) suggested that interaction between milk proteins and chocolate avonoids inhibits the absorption of ()-epicatechin (()-EC)
into the bloodstream. On the other hand, Schroeter et al. (2003)
showed that there was no signicant difference in the ()-EC concentration in the plasma after the consumption of a milk-containing
or water-based cocoa beverage under isocaloric and isolipidemic
conditions. Schramm et al. (2003), after evaluating the effect of several foods including sugar, milk, bread, steak, and grapefruit on the absorption and pharmacokinetics of cocoa avanols, also concluded that
milk had no effects on avanol absorption.
4.4. Effects on in-vitro bioavailability
There are several studies which investigated the effect of protein
phenolic compound interactions on in vitro bioavailability. Dupas et
al. (2006) studied the effect of milk addition to coffee on the bioavailability of coffee phenolics, mainly CGA. Interactions between CGA and
milk proteins were investigated using an ultraltration technique.
These interactions proved to be slightly disrupted during an in vitro
digestion process. CGA absorption and bioavailability were then studied in vitro using a Caco-2 cell model coupled with an in vitro digestion process. The results revealed that CGA absorption under its
native form is weak, and unmodied by the addition of milk proteins,
but slightly reduced by the addition of Maillard reaction products.
These data showed the presence of interactions between coffee phenolics and milk proteins, but there was no signicant effect observed
on the bioavailability of CGA (Dupas et al., 2006). On the other hand,
Neilson et al. (2009) studied the effect of milk addition to chocolate
on the bioavailability of EC of chocolate. According to the results, in

vitro bioaccessibility and Caco-2 accumulation did not differ between
treatments. Bioavailability as measured by the areas under the serum
concentrationtime curve (AUC) was not signicantly different
between confections which is in agreement with the studies
suggesting that the presence of milk does not negatively affect the
bioavailability of cocoa avan-3-ols (Neilson et al., 2009).
Arimboor and Arumughan (2011) studied the effect of sea buckthorn proanthocyanidins on in vitro digestion of proteins. It was
found that interactions of sea buckthorn proanthocyanidins with
food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailability, negatively. In another study,
Nizamova et al. (2011) proposed a new method for the estimation
of the bioavailability of polyphenols using electrogenerated bromine
as a coulometric titrant. The titration of model solutions of casein
and BSA showed that casein did not interact with electrogenerated
bromine, while BSA reacted with the titrant in the ratio of 1:63. The
proteins bound rutin and quercetin at a high rate and thus reduced
the bioavailability of polyphenols. The concentration of free polyphenol was reduced with an increase in the concentration of protein in
the mixture. The total antioxidant capacity of tea samples was also
determined, and it was reported that green tea possess higher total
antioxidant capacity than the black one because of the partial oxidation of polyphenols to respective thearubigins and theaavins at the
fermentation step in the production of green tea. According to the results, the total antioxidant capacity of tea decreased with the amount
of milk. Milk proteins bound tea polyphenols into complexes as a result of intermolecular interactions and reduced their bioavailability,
accordingly (Nizamova et al., 2011).
5. Discussion
As a result of proteinphenolic interactions changes occur both on
the proteins and phenolic compounds. The main changes observed in
proteins are basically related with the structure, functional and nutritional properties, and digestibility of proteins. On the other hand,
these interactions result with changes in total phenolic or avonoid
contents, antioxidant activities, contents of individual phenolic compounds, as well as in-vitro and in-vivo bioavailability of phenolic
compounds. Conditions such as temperature, pH, types of proteins,
protein concentration, types and structures of phenolic compounds
and several other factors affect proteinphenolic interactions. There
are some contradictory results about the effect of temperature on
the binding afnity of polyphenols to proteins. Some studies indicated that binding afnity decreases by increasing temperature whereas
some others reported vice versa. Unlike the temperature, there
are more comparable results regarding the effects of pH on protein
phenolic interactions. Lower pH results with a stronger binding
since the dissociation of protein leads to more binding sites at lower
pH values. On the other hand, the different hydrophobicity and isoelectric points of proteins, and the difference in the amino acid composition lead to differences in the binding afnity of phenolic
compounds to some proteins. The protein concentration in the solution
affects the proteinphenolic interactions, thus the precipitation is decreased after high concentrations reached in the solution. Different
types and structures of phenolic compounds also affect proteinphenolic interactions. Some of the structural elements that inuence the afnities of polyphenols for proteins are the following (Xiao et al., 2011): (i)
Methylation and methoxylation of avonoids decreased or only slightly
affected the afnities; (ii) hydroxylation on rings A and B of avones
and avonols slightly enhanced the interaction and hydroxylation on
the ring A of avanones and signicantly improved the afnities; (iii)
glycosylation of polyphenols weakened the afnities; (iv) hydrogenation of the C2_C3 double bond of avonoids decreased the binding
afnities (v) galloylation of catechins and esterication of gallic acid
signicantly improved the binding afnities. There are also other factors
967
that inuence the proteinphenolic interactions such as salt concentration and addition of certain reagents. However, the number of studies
investigating the effect of all these parameters is very limited and
should be further studied in more detail to optimize the process conditions and to improve the benecial health effects of phenolics. Process
conditions and products can then be designed in a way that will provide
the maximum benecial health effects to the consumers, and also optimum nutritional and functional properties can be supplied to the
products.
Proteinphenolic interactions inuence the structure, functional
and nutritional properties, and digestibility of proteins. It was observed that the proteinphenolic interactions increase the molecular
weight of proteins. The presence of phenolic compounds also affects
protein solubility which is an important factor in protein functionality, because protein insolubility also hinders other protein functional
properties. The increase in the content of phenolic compounds decreases the solubility of proteins. In addition, interaction of proteins
with phenolic compounds may improve the thermal stability of proteins. Moreover, proteins may also lead to some undesirable changes
in color and taste when they interact with phenolic compounds. Furthermore, looking from the nutritional value and digestibility point of
view, it was observed that proteinphenolic interactions decrease the
nutritional value of some essential food components and also in vivo
and in vitro digestibility of proteins.
On the other hand, some other studies focused on the changes in
phenolic compounds as a result of proteinphenolic compound interactions and investigations on the changes in antioxidant capacities and
bioavailability of phenolic compounds were performed. However, research on individual phenolic compounds is lacking. On the other
hand, although the bioactivity of a molecule mainly depends on the
functional group/moiety in the molecule, the differences in the molecular structure were not considered in the studies performed so far. It is
important to understand the involvement of the functional groups or
the position of these functional groups in proteinphenolic complex,
and it should be investigated in detail to better understand the mechanisms taking place.
Another important drawback of the proteinphenolic interaction
studies is that they are generally performed using only tea, coffee,
and chocolate as phenolic compound sources and milk as the protein
source. Tea and coffee are the most widely consumed beverages in
the world with a high antioxidant capacity and they are often consumed together with milk. Therefore, it is important to understand
the effect of milk proteins on the antioxidant capacity and bioavailability of coffee and tea phenolics, as they also account for most of
the polyphenol daily intake in the countries where the fruits and vegetables are not preferred to be eaten very often. However, it is still of
critical importance to work on other food proteins such as plantoriginated proteins, to understand the effects more clearly and to be
able to compare the effect of different protein sources on these interactions. On the other hand, a better understanding of the protein
phenolic interactions for a large variety of food products may provide
benet for improving the process conditions and parameters for the
food products that contain both proteins and phenolic compounds.
This information can also help developing new food products with
better nutritional quality and improved health aspects.
According to many of the studies reviewed in this paper, it can be
concluded that proteins signicantly decrease the antioxidant capacity in general. There are some contradictory results between these
studies which may arise from different methodologies used to measure the antioxidant capacity or total phenolic/avonoid contents. It
is expected that a single method cannot determine all the antioxidant
compounds available in the food matrix correctly. All the methods
have their own advantages and disadvantages. Even the results of
the methods sharing the same principle like ABTS and DPPH can
show important differences in their response to antioxidants. Similarly, the extraction procedure used during analysis needs special
968
attention as the stability of proteinphenolic complex might be different in different solvent systems. As mentioned above, there are
few studies about the effect of phenolic-protein interactions on individual phenolics. Most of the studies measured the total content of
phenolics, and avonoids or the total antioxidant capacity, but there
is still a need for more studies that investigate individual phenolics
to understand the mechanism better. Especially, use of more comprehensive methods such as LCMS will provide more detailed information on the interactions of proteins and phenolic compounds.
Contradictory results are observed also in the bioavailability studies. Some studies indicated that proteinphenolic compound interaction decreases the bioavailability, others reported no signicant
changes and a very few revealed that it increases the bioavailability
of some phenolic compounds. This fact might be due to a high variability in the absorption of avanols in humans, as well as to the
small number of subjects selected in the studies. Thus, bioavailability
of phenolics should be studied in a homogenous population including
a greater number of subjects.
It is also of critical importance to evaluate the bioavailability of
health associated compounds present in these food materials, which
will provide valuable data for elucidating the true biological relevance
of these compounds in the context of nutrition and human health.
Bioavailability differs greatly from one polyphenol to another, and
for some compounds it depends on the dietary source. So, it is important to work with different food sources of proteins and phenolic
compounds to better understand the exact effect of interactions of
these two compounds. According to the results of some bioavailability studies, in vitro methods can be well correlated with the results of
in vivo experiments (Bouayed, Hoffman, & Bohn, 2011). In vitro digestion and dialysis methods for simulating the gastrointestinal (GI)
digestion are being extensively used since they are rapid, safe, and
do not have the same ethical restrictions as in vivo methods (Liang
et al., 2012). On the other hand, some researchers reported that the
results of in vitro methods cannot be fully and directly extended to
the results of in vivo methods (Green et al., 2007). So, the interaction
of proteins and phenolic compounds would be better evaluated by
both in vitro and in vivo models to provide comparable and more accurate results.
6. Conclusions
In summary, polyphenols which have been widely studied for
their health promoting and disease preventive activities in humans
are well-known to have high afnity to bind proteins. Therefore, interaction of phenolics and proteins affect both of these food constituents in
food systems. However, the mechanisms and the consequences of their
interactions should be studied extensively due to the fact that contradictory results were obtained so far. Although, there are several studies
investigating the effect of proteinphenolic interactions on the antioxidant capacities and bioavailability of phenolic compounds, research on
individual phenolic compounds is lacking. On the other hand, understanding the exact mechanism for a large variety of food products
may provide benets for improving the process conditions and parameters for the food products that contain both proteins and phenolic compounds. Outcomes of these studies can help devolop new
food products with better nutritional quality and benecial health
aspects.
It can be concluded that proteins signicantly decrease the antioxidant capacity in general, but there are some controversies about the
results observed by different antioxidant capacity methods which is
probably due to the differences in the principles or fundamentals of
these methods. That's why it is recommended to use several test
methods and compare the results obtained. In addition, more comprehensive methods such as LCMS would better be used to obtain
more detailed information on the interactions of proteins and phenolic compounds. Results of bioavailability studies can be strengthened
by using a homogenous population including a greater number of

subjects. Further work on this topic is still necessary to clarify the
controversial results obtained so far and to better understand the
mechanisms underlying proteinphenolic interactions as well as factors affecting the degree of this interaction.
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Protein Phenolic Interaction

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Food Research International 51 (2013) 954970

Contents lists available at SciVerse ScienceDirect

Food Research International

A review on proteinphenolic interactions and associated changes

T. Ozdal et al. / Food Research International 51 (2013) 954970

T. Ozdal et al. / Food Research International 51 (2013) 954970

Fig. 2. The structure of various phenolic compounds.

as repulsive component. Depending on the relative number of negatively

during processing, transportation and storage. In this review, the effects

T. Ozdal et al. / Food Research International 51 (2013) 954970

T. Ozdal et al. / Food Research International 51 (2013) 954970

Van der Waals

Green tea avonoids Number of surface

The structural stabilization of

FTIR, CD, uorescence

Circular dichroism, differential

Texture prole analysis, color

Gallic acid and rutin

Yan, Li, Zhao,

Heat coagulation time-pH

Tannic acid, caffeic

1) Mechanical strength of gels

Texture prole analysis,

Mechanical properties, water

T. Ozdal et al. / Food Research International 51 (2013) 954970

2.3. Types of proteins and protein concentration

weakens the afnity for proteins. Glycosylation of resveratrol slightly

2.4. Types and structures of phenolic compounds

Other factors inuencing the proteinphenolic interactions are

T. Ozdal et al. / Food Research International 51 (2013) 954970

with LG in solution. The order of binding increases as the

Walnut kernels have a signicant amount of phenolic compounds,

T. Ozdal et al. / Food Research International 51 (2013) 954970

A transfer in isoelectric points of soy protein to more acidic pH

3.3. Effects on nutritional value and digestibility

4. Effects of proteinphenolic compounds interactions on

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Almajano et al. (2007) mixed BSA, -lactoglobulin, -lactalbumin,

Arts et al. (2002)

Whole kernel b hull

Ryan and Petit

Black tea phenolics

Ryan and Sutherland

Milk proteins and

Epigallocatechin gallate (EGCG)

Dairy products &

Cocoa liquor, cocoa powder,

Dairy products & tea Milk proteins

Tea polyphenols (Green tea,

Dairy products & tea Milk proteins

Tea polyphenols (Green and

Argentinean black tea

Espresso, Turkish, Instant

Coffee & milk

Coffee & tea & milk

Tea and coffee

Dubeau et al. (2010)

T. Ozdal et al. / Food Research International 51 (2013) 954970

Studies investigating the effect of milk on antioxidant capacities of

effects of milk addition. The results revealed that in comparison to

T. Ozdal et al. / Food Research International 51 (2013) 954970

acid, protocatechuic acid, caffeic acid and (+)-catechin), the strongest

volunteers who consumed either 200 g of blueberries plus 200 ml

T. Ozdal et al. / Food Research International 51 (2013) 954970