Food Research International 51 (2013) 954–970

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

A review on protein–phenolic interactions and associated changes

Tugba Ozdal a, Esra Capanoglu b,⁎, Filiz Altay b
a
Department of Food Engineering, Faculty of Engineering and Architecture, Okan University, Tuzla, TR-34959, Istanbul, Turkey
b
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 a b s t r a c t

Article history: Polyphenols have become an intense focus of research interest due to their health-beneficial effects especially in the
Received 1 August 2012 treatment and prevention of several chronic diseases. Polyphenols are known to form complexes with proteins lead-
Accepted 9 February 2013 ing to changes in the structural, functional and nutritional properties of both compounds. In this review, the effects
of protein–phenolic interactions under various conditions on protein and phenolic compound's structure and func-
Keywords:
tionality are described. The parameters that are defined to affect protein–phenolic interactions are basically temper-
Protein–phenolic interactions
Proteins
ature, pH, protein type and concentration, and the type and structure of phenolic compounds. Even though the exact
Phenolics mechanism of how proteins influence polyphenols is still not yet known, studies on the changes in the structure and
Total antioxidant capacity functional properties were investigated. According to these studies, secondary and tertiary structures of the proteins
Bioavailability 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 significantly 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 com-
pounds 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 beneficial health effects to the con-
sumers with optimum nutritional and functional properties.
© 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
2. Parameters affecting interactions between protein and phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956
2.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956
2.2. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957
2.3. Types of proteins and protein concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
2.4. Types and structures of phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
2.5. Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
3. Effects of protein–phenolic compounds interactions on proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
3.1. Effects on structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
3.2. Effects on functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 960
3.3. Effects on nutritional value and digestibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961
4. Effects of protein–phenolic compounds interactions on phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961
4.1. Effects on total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity . . . . . . . . . . . . . . . . . . . . 961
4.2. Effects on the content of individual phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963
4.3. Effects on in-vivo bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 964
4.4. Effects on in-vitro bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967

⁎ 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: [email protected] (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) 954–970 955

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968

1. Introduction Zhang, 2011), vasodilatory (Mudnic et al., 2010), and analgesic activ-
ities (Santoz, Almeida, Lopez, & Souza, 2010).
Proteins are highly complex polymers, made up of twenty different Polyphenols can be oxidized by molecular oxygen with side chain
amino acids consisting of an α-carbon atom covalently attached to a hy- amino groups of peptides at alkaline pH to quinines, leading to the for-
drogen atom, an amino group, a carboxyl group, and a side-chain R mation of protein cross-links (Damodaran, 1996; Prodpran, Benjakul, &
group (Fig. 1) (Damodaran, 1996). The differences in structure and Phatcharat, 2012). These highly reactive quinines can irreversibly react
function of proteins arise from the sequence in which the amino acids with the sulfhydryl and amino groups of proteins. In addition, quinines
are linked together via amide bonds. Proteins are important food com- can undergo condensation reactions, resulting in the formation of high
ponents existing mostly in milk, meats (including fish and poultry), molecular weight brown colored pigments named as tannins. Tannins
eggs, cereals, legumes and oilseeds. They can form complexes with are highly reactive and can readily combine with SH and amino groups
other food components including polyphenols leading to changes in of proteins. Quinone–amino group reactions are known to decrease the
their structural, functional and nutritional properties. A fundamental digestibility and bioavailability of protein-bound lysine and cysteine
understanding of these changes as a result of interactions with phenolic (Damodaran, 1996).
compounds is essential from the scientific, industrial, and economical The phenolic group is an excellent hydrogen donor that forms hy-
point of view. drogen bonds with the carboxyl group of the protein. For phenolic
Phenolic compounds are chemically structured as a hydroxyl group compounds to have high protein affinity, they must be small enough
bonded to an aromatic ring (Fig. 2). They are secondary metabolites, not to penetrate inter-fibrillar regions of protein molecules, but large
involved in growth and energy metabolism in the body (Harnly, enough to crosslink peptide chains at more than one point (Mulaudzi,
Bhagwat, & Lin, 2007). There are currently more than 8000 known phe- Ndhlala, Kulkarni, & Staden, 2012). The molecular explanations of pro-
nolic compounds identified in fruits, vegetables, seeds, and liquids tein–phenolic interaction are given in Fig. 3. The diphenol moiety of a
(Cuykens & Claeys, 2004; Guo, Kong, & Meydani, 2009). Phenolic com- polyphenol (Almajano, Delgado, & Gordon, 2007) is readily oxidized
pounds can be classified into two groups: basic phenolic compounds to an ortoquinone, either enzymatically as in plant tissues, or by molec-
and polyphenols (Vermerris & Nicholson, 2006). Dietary polyphenols ular oxygen (Damodaran, 1996; Strauss & Gibson, 2004). The quinine
represent the main source of antioxidants for human use (Graf, forms a dimer (Arimboor & Arumughan, 2011) in a side reaction, or re-
Milbury, & Blumberg, 2005). The main classes of polyphenols are de- acts with amino or sulfhydryl side chains of polypeptides to form cova-
fined according to the nature of their carbon skeleton: phenolic acids, lent C\N or C\S bonds with the phenolic ring, with regeneration of
flavonoids, and the less common stilbenes and lignans. Phenolic acids hydroquinone. The latter can be reoxized and bind a second polypep-
include caffeic acid, ferulic acid and hydrolyzable tannins. Flavonoids tide, resulting in a cross-link (Arts, Haenen, Voss, & Bast, 2001). Other-
can be divided into several classes according to their degree of oxidation wise, two quinines, each carrying one chain, can dimerize, producing
in the heterocycle (Guo, Kong, & Meydani, 2009). Flavonoids include a cross-link as well (Arts et al., 2002) (Strauss & Gibson, 2004).
flavonols (e.g., quercetin and kaempferol, the most ubiquitous flavonoids The interactions of phenolic compounds with proteins may lead to
in foods), flavones, isoflavones, flavanones, anthocyanins, flavanols changes in physico-chemical properties of proteins such as solubility,
(catechins-monomers and proanthocyanidin polymers, known as con- thermal stability, and digestibility (Labuckas, Maestri, Perelló, Martínez,
densed tannins) (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004; & Lamarque, 2008; Rawel, Kroll, & Rohn, 2001). Additionally nutritional
Scalbert & Williamson, 2000). properties of proteins may be affected due to the modification of essen-
Polyphenols have become an intense focus of research interest tial amino acids and through the inhibition of proteases (Kroll, Rawel, &
due to their health-beneficial effects especially in the treatment and Rohn, 2003). On the other hand, the interactions of other compounds in-
prevention of cancer (Chen et al., 2011; Weng & Yen, 2012) and car- cluding lipids (Smith, 2012), other proteins (Hsu, Pang, Sheetal, &
diovascular diseases (Kuriyama et al., 2006; Mursu et al., 2008). The Wilkins, 2007; Thangudu, Bryant, Panchenko, & Madej, 2012), vitamins
suggested beneficial effects include anticarcinogenic (Jeong et al., (Relkin & Shukat, 2012) with proteins are also known to change the
2011; Ogunleye, Xue, & Michels, 2009), antiatherogenic (Liu, Zubik, characteristics of those compounds. Polyphenols may interact with pro-
Collins, Marko, & Meydani, 2004; Mulvihill & Huff, 2010), antiulcer teins both reversibly and irreversibly. Some of the examples from both
(Zakaria et al., 2011), antithrombotic (Han et al., 2012; Tao et al., interactions in the literature are given in Table 1. In reversible interac-
2012), anti-inflammatory (Beara et al., 2012; Zimmer et al., 2012), tions, usually non-covalent forces such as hydrogen bonding, hydropho-
antiallergenic (Chung & Champagne, 2009; Schmitz-Eiberger & bic bonding and van der Waals forces are involved (Charlton et al., 2002;
Blanke, 2012), anticoagulant (Bijak et al., 2011), immune modulating Jobstl, O'Connell, Fairclough, & Williamson, 2004; Poncet-Legrand et al.,
(Schütz, Saß, With, Graubaum, & Grünwald, 2010), antimicrobial 2006; Prigent, Gruppen, Visser, Van Koningsveld, & Alfons, 2003;
(Silva, Rodrigues, Feas, & Estevinho, 2012; Xia, Wu, Shi, Yang, & Richard, Lefeuvre, Descendit, Quideau, & Monti, 2006; Richard, Vitrac,
Merillon, & Monti, 2005; Siebert, 2006), whereas in irreversible interac-
tions, covalent bonds are formed between the polyphenols and proteins
COOH (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 interac-
tion. They are only stable as long as they are protected from water. Van
H C NH2 der Waals interactions are intermolecular interactions affected by the
surrounding solvent. They are dipole–induced dipole and induced-
dipole–induced dipole interactions between neutral atoms in protein
R molecules. When two atoms come close to each other, each atom
induces a dipole in the other via polarization of the electron cloud. The
Fig. 1. The structure of an amino acid. interactions between these induced dipoles have an attractive as well
956 T. Ozdal et al. / Food Research International 51 (2013) 954–970

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
and positively charged residues, proteins have either a net negative or a of protein–phenolic interactions under various conditions on protein
net positive charge at neutral pH. These charged groups in proteins and phenolic compounds' structure and functionality are described.
are distributed on the surface of the protein molecule. Electrostatic
interactions occur between like charges (repulsive interactions) and 2. Parameters affecting interactions between protein and
opposite charges (attractive interactions), which are charged groups on phenolic compounds
the surface of the protein molecule and the other molecules in the
media. Hydrogen bonding and electrostatic interactions between There are many parameters that affect protein–phenolic interac-
various polar groups are not very stable, and their stabilities depend on tions such as temperature, pH, types of proteins, protein concentration,
maintenance of an apolar environment. Hydrophobic interactions types and structures of phenolic compounds, salt concentration, and addi-
among nonpolar groups are stronger (Damodaran, 1996). The number tion of certain reagents. Complex formation of protein and phenolics re-
of studies which describe the reversible interactions is more than sults from hydrogen binding and hydrophobic interactions (Hagerman
the irreversible ones mainly due to the lack of suitable methods for & Klucher, 1986). Specific to protein–phenolic complex, hydrophobic in-
quantitating the covalent bonds between molecules. teractions have been considered to be promoted by hydrogen bonding
Trombley, Loegel, Danielson, and Hagerman (2011) have suggested (Haslam, 1996), as summarized in Table 1.
that bioactivities and bioavailability of plant polyphenols and other cat-
echin derivatives may be affected by the covalent interaction between 2.1. Temperature
polyphenols and proteins. Reaction mechanisms and methods for char-
acterizing non-covalent and covalent interactions between polyphenols Temperature can affect hydrogen bondings and causes the formation
and proteins were reviewed thoroughly by Bourvellec and Renard of hydrophobic bondings, therefore, it is an important parameter in the
(2012), very recently. protein–phenolic interactions. Sastry and Rao (1990) observed that tem-
The interactions of phenolic compounds and proteins are known to perature had a significant influence on the binding of polyphenol-free
affect the structure of proteins, content of free polyphenols, antioxidant 11S protein of sunflower seed with 5-O-caffeoylquinic acid. The binding
capacity and bioavailability of phenolic compounds in foods. A better of polyphenol-free 11S protein of sunflower seed and 5-O-caffeoylquinic
understanding of phenolic compound–protein interactions would acid significantly decreased as the temperature increased from 30 °C to
help to control the functional properties of proteins in food products 45 °C and it completely disappeared at 55 °C. Temperature affected
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 957

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 affinity of point of the proteins (Naczk, Grant, Zadernowski, & Barre, 2006). Un-
5-O-caffeoylquinic acid to polyphenol-free 11S protein as a result of the like the temperature, pH affected only the degree of binding but not
important role of hydrogen bonding in the binding of 5-O-caffeoylquinic the binding affinity for the interaction between polyphenol-free 11S
acid by the 11S protein (Sastry & Rao, 1990). Prigent et al. (2003) studied protein of sunflower seed and 5-O-caffeoylquinic acid. Lower pH led
interactions of bovine serum albumin (BSA) with 5-O-caffeoylquinic acid to stronger binding because the dissociation of protein had more
at 5, 25 and 60 °C. They reported that the binding affinity of 5-O- binding sites at lower pH (Sastry & Rao, 1990). The interactions be-
caffeoylquinic acid for BSA decreased with temperature. However, tween chlorogenic acid (CGA) and several proteins such as BSA, lyso-
Hoffmann et al. (2006) reported that precipitation of BSA with zyme, and α-lactalbumin at pH ≤ 7 produced non-covalent bonds
procyanidin derivatives was independent of temperature variations. and the amount of CGA bound by BSA (per molecule) was somewhat
Tsai and She (2006) evaluated the contribution of phenol–protein higher at lower pH (Prigent et al., 2003). With the increase in pH, the
interactions on the antioxidant capacity of peas after immersion covalent interaction between lysozyme and CGA was stronger due to
with five phenolics under different heating conditions between 30 the formation of more radicals or quinones from the autoxidation of
and 70 °C. They extracted superoxide dismutase (SOD) enzyme CGA at higher pH. The reactive radicals and quinones subsequently
from peas, formed protein–phenolic interaction complex and measured interacted with proteins covalently (Prigent et al., 2003).
SOD activity and binding capacity of this complex with pea protein. SOD Naczk, Oickle, Pink, and Shahidi (1996) studied the effect of pH on
activity found in fresh or processed fruits is very weak as a result of the formation of crude tannin canola extract/BSA, fetuin, gelatine, and
protein deformation induced by heating time and temperature. They lysozyme complexes. It was suggested that the optimal pH for precipi-
reported that the heat stability of SOD increased after interacting with tation varies for different proteins and generally it is close to the isoelec-
phenolic compounds as a result of the increase in SOD activation energy tric point of the protein. Rawel, Meidtner, and Kroll (2005) also
caused by the binding of phenolic compounds to protein. It was ob- observed higher binding affinity for ferulic acid and CGA close to the iso-
served that the binding effect of phenolic compound was increased electric point of BSA. Frazier, Papadopoulou, and Green (2006) failed to
with temperature (Tsai & She, 2006). find an effect of pH on binding of (−)-epicatechin to BSA. This was also
supported by results of Papadopoulou, Green, and Frazier (2005) and
2.2. pH Charlton et al. (2002) and suggested that electrostatic interactions are
not a major factor in forming the (−)-epicatechin/BSA complex. Indeed,
The precipitations of complexes resulting from polyphenol–protein increased precipitation of protein/polyphenol complexes close to the
interactions are pH sensitive. The lowest solubility of polyphenol– isoelectric point may be attributed to the minimum solubility of the
protein complexes occurred at 0.3–3.1 pH units below the isoelectric protein at this pH.
958 T. Ozdal et al. / Food Research International 51 (2013) 954–970

Table 1
Different types and mechanisms of protein–phenolic interactions.

Type of interaction Interaction Protein Phenolic compound Changes in functionality or Assessment method References
mechanism modification

Reversibly Non-covalent Hydrogen bonding α-lactalbumin Procyanidins of Procyanidins of medium DP Isothermal titration Prigent et al.,
forces lysozyme various degree of can lead to an undesirable calorimetry 2009
BSA polymerization (DP) decrease of protein solubility,
but may play a positive role
in foam stability
Hydrogen bonding Bovine serum Ferulic acid (FA) Thermal stability of BSA fluorescence, circular Ojha, Mishra,
albumin (BSA) increases upon binding with dichroism and isothermal Hassan, and
FA. titration calorimetry Chaudhury
(2012)
Van der Waals Bovine (−)-epigallocatechin contribute to the fluorescence spectra, CD Wu et al.
forces + Hydrogen β-lactoglobulin functionality of the milk spectra, infrared spectroscopy, (2011)
bonding products and synchronous
fluorescence spectra
Hydrophobic β-casein in milk Green tea flavonoids Number of surface Fluorometry analysis, Yüksel et al.
binding (catechins) hydrophobic sites decreased isothermal titration (2010)
with phenolics calorimetry
Hydrophobic Walnut proteins Walnut phenolics The presence of phenolic SDS-PAGE Labuckas et
binding compounds decreased protein al. (2008)
solubility in walnut flour
obtained from whole kernels.
Hydrogen bonding Sorghum Sorghum tannins Precipitation of proteins, and – Duodu,
and non-polar proteins make them insoluble and Taylor,
hydrophobic indigestible Belton, and
interactions Hamaker
(2003)
Hydrophobic and Milk Tea polyphenols The structural stabilization of FTIR, CD, fluorescence Kanakis et al.
hydrophilic β-lactoglubulin protein increased spectroscopic methods (2011)
interactions
Irreversibly Covalent Soy protein Chlorogenic-, caffeic-, 1) Reduction in lysine, Circular dichroism, differential Rawel,
bonds gallic acid, flavones, cysteine and tryptophan, 2) scanning calorimetry (DSC) Czajka, Rohn,
apigenine, The isoelectric points shifted and Kroll
kaempferol, to lower pH, 3) Increase in (2002)
quercetin and molecular weight, 4) More
myricetin hydrophilic surface on soy
protein, 5) Influence on
solubility
Fish myofibrillar Caffeic acid, 1) Enhanced mechanical Texture profile analysis, color Prodpran et
protein catechin, ferullic properties with phenolic measurement, light al. (2012)
acid and tannic acid compounds, 2) Influence on transmission, SDS-PAGE
properties and appearance of
protein films
Gelatin Gallic acid and rutin 1) Increased gel strength, Texture profile analysis, Yan, Li, Zhao,
thermal stability, 2) Decrease rheometry, DSC, swelling tests, and Yi (2011)
in swelling scanning electron microscopy,
X-ray diffraction, FTIR
Non-disulphide β-lactoglobulin Sour cherry 1) Allergenicity of protein SDS-PAGE, isoelectrofocusing, Tantoush et
covalent linkages phenolics decreased, 2) Digestibility immunoblotting, al. (2011)
(antocyanins) remained size-exclusion and
reverse-phase chromatography,
mass spectrometry, digestibility,
antioxidant activity
Cross-linking Gelatin (Type A) Phenolic acid, 1) Mechanical strength of gels Free amino groups analysis, Strauss and
quercetin, rutin increased, 2) Reduced swelling, gel rigidity, swelling, dynamic Gibson
3) Fewer free amino groups, light scattering (2004)
4) denser polymeric networks
Milk protein Caffeic acid 1) Enhance the heat stability of Heat coagulation time-pH O'Connell
milk profile, determination of and Fox
2) Reduced the lysine and available lysine, sulfhydryl (1999)
sulfhydryl content of heated milk groups, zeta potential, relative
3) No effect on rennet viscosity, measurement of
coagulation time, alcohol casein micelle size
stability, viscosity or
zeta potential
4) Increased casein micelle size
5) Increased crosslinking of milk
proteins
Cross-linking Porcine plasma Tannic acid, caffeic 1) Tensile strength increased, Mechanical properties, water Nuthong,
protein acid, ferulic acid 2) Elongation at break vapor permeability Benjakul, and
increased, 3) water vapor Prodpran
permeability of films increased (2009)
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 959

2.3. Types of proteins and protein concentration weakens the affinity for proteins. Glycosylation of resveratrol slightly
reduced the affinity for milk proteins. The affinity of resveratrol for
Protein–phenolic interaction is affected by the types of proteins and milk proteins was about 2.51 times higher than that of polydatin. The
the molar ratio of phenolic/protein (Prigent et al., 2003). They can bind hydrogenation of the C2_C3 double bond of flavonoids decreased the
either hydrophobically or hydrophilically depending on binding sites of binding affinities for milk proteins about 7.24 to 75.86 times. The affin-
the protein. The difference in binding affinity among proteins depends ities of apigenin and myricetin for milk proteins were about 75.86 times
on several factors, such as hydrophobicity (BSA>α-lactalbumin > and 8.51 times higher than those of naringenin and dihydromyricetin,
lysozyme), isoelectric point and the amino acid composition of proteins respectively. The hydrogenation of the C2_C3 double bond for many
(Prigent et al., 2003). For example, the binding affinity of CGA to BSA was flavonoids decreased the binding affinity for BSA by 2–4 orders of mag-
higher than to lysozyme and α-lactalbumin. The amount of protein in nitude. Galloylation of catechins significantly improved the binding af-
the solution affects the protein–phenolic interactions as well. If the con- finities with milk proteins about 100–1000 times. The pyrogallol-type
centration of BSA was low, the difference of protein precipitation catechins showed lower affinities than catechol-type catechins. More-
between 0.5 mg/ml of BSA and 1.0 mg/ml of BSA was not statistically over, the affinity of catechin with 2,3-trans structure for milk proteins
significant. On the other hand, when the concentration of BSA was was found to be higher than that of the catechin with 2,3-cis structure.
higher than 1.0 mg/ml, the protein precipitation effect was significantly The esterification of gallic acid significantly improved the affinity for milk
lower comparing to the effect obtained from higher concentrations of proteins. The affinities of gallic acid and its esters with α-amylase were
BSA (Naczk et al., 1996). 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 protein–phenolic inter-
actions depending on factors such as molecular weight, methylation, hy- Other factors influencing the protein–phenolic interactions are
droxylation, glycosylation, and hydrogenation of phenolic compounds. salt concentration and addition of certain reagents. The binding
The binding affinity of polyphenols to proteins increases with their mo- strength of CGA to sunflower 11S protein was reduced with an in-
lecular size. Larger polyphenols like those present in black tea (theaflavin, crease in NaCl concentration. The concentration of NaCl lowered the
thearubigin) are more likely to bind milk proteins due to the fermentative amount of binding points instead of affecting the binding affinity
oxidation/polymerization of catechin monomers (Dubeau, Samson, & due to fact that salts at high concentration can inhibit the dissociation
Tajmir-Riahi, 2010). Among several low molecular weight phenolic com- of oligomeric proteins (Sastry & Rao, 1990). Some reagents, such as
pounds including p-coumaric acid, p-hydroxybenzoic acid, cinnamic acids Na2SO3 (a reducing agent), even at low concentration can affect the
(protocatechuic acid and caffeic acid) and catechin, the 3,4-dihydroxy protein–phenolic interactions, the binding between CGA and 11S pro-
benzoic and cinnamic acids had the strongest binding affinity for BSA tein disappeared completely in 0.01 M Na2SO3 (Sastry & Rao, 1990).
while there was no significant interaction between p-hydroxybenzoic
acid and BSA (Bartolome, Estrella, & Hernandez, 2000). Although both 3. Effects of protein–phenolic compounds interactions on proteins
quercetin and quercetin 3-O-β-D-glucopyranoside are flavonoids, their
binding affinities with BSA were different since stronger interaction was 3.1. Effects on structure
observed between BSA and quercetin (Martini, Claudia, & Claudio,
2008). For increasing the heat stability of SOD in peas, hydroxycinnamic The mechanism of how proteins influence polyphenols is still not
acids including ferulic acid, coumaric acid and caffeic acid were found to yet known. In order to give an explanation, firstly the changes in the
increase the heat stability better than hydroxybenzoic acid; coumaric structures of the proteins should be well understood. There are sever-
acid was found to be superior for enhancing the antioxidant activity of al researches that have been performed recently to understand the
SOD and showed the strongest binding ability with pea protein (Tsai & mechanism by which the antioxidants in tea are affected by the addi-
She, 2006). tion of milk.
Xiao et al. (2011) investigated the relationship between the struc- Hasni et al. (2011) studied the interaction of α- and β-caseins
tural properties of dietary polyphenols and their affinities for milk with tea polyphenols (+)-catechin (C), (+)-epicatechin (EC),
proteins. Methylation and methoxylation of flavonoids weakened or (+)-epigallocatechin (EGC) and (+)-epigallocatechin gallate (EGCG)
little affected their binding affinities for milk proteins. In general, at a molecular level, using Fourier transform infrared (FTIR), UV–visible,
the methylation of hydroxyl group in flavonoids decreased their bind- circular dichroism (CD), fluorescence spectroscopic methods and mo-
ing affinities for milk proteins by 1.10–14.79 times. The affinity of lecular modeling. It was concluded that tea polyphenols weakly bind
daidzein for milk proteins was found to be 14.79-times higher than to α-casein and β-casein through both hydrophilic and hydrophobic in-
that of its methylated form (formononetin). 40-methoxylation of teractions. The order of binding increases as the number of OH group in-
galangin hardly affected the affinity for milk proteins. Hydroxylation creased with C ~ EC > EGC > EGCG. β-Casein forms stronger complexes
on the rings A and B of flavones and flavonols slightly enhanced the with tea polyphenols than α-casein, due to the more hydrophobic
binding affinities for milk proteins. The hydroxylation on the ring A nature of β-casein. Structural modeling of the interaction between α-
of flavanones significantly improved the affinities for milk proteins. and β-caseins and tea polyphenols showed that the participation of
However, the hydroxylation on the ring C of flavones hardly influenced several amino acid residues in polyphenol–protein complexation with
the binding affinities for milk proteins and the hydroxylation on ring A extended H-bonding network. Casein conformation was changed by
of isoflavones reduced or little affected the affinities for milk proteins. polyphenol with a major reduction of α-helix and β-sheet and increase
Quercetin binding affinities for milk proteins was found to be only of random coil (Hasni et al., 2011).
1.02 times higher than that of quercitrin which is the glycoside formed Kanakis et al. (2011) investigated the interaction of β-lactogolobulin
from quercetin. The affinities (logKa) of naringenin, naringin and (βLG) with tea polyphenols (+)-C, (−)-EC, (−)-ECG and (−)-EGCG at
narirutin for milk proteins were determined as 3.94, 3.76, and 3.64, re- molecular level, using FTIR, CD, fluorescence spectroscopic methods
spectively. It revealed that the monoglycosides of flavonoids showed and molecular modeling. They determined polyphenol binding mode,
stronger binding affinities with milk proteins than their polyglycoside the binding constant and the effects of polyphenol complexation on
forms. The decreasing affinity for milk protein after glycosylation may βLG stability and secondary structure. As a result of structural analysis
be caused by the non-planar structure. After the hydroxyl group is it was observed that polyphenols bind βLG through both hydrophilic
substituted by a glycoside, steric hindrance may take place, which and hydrophobic interactions. Tea polyphenols make weak bonds
960 T. Ozdal et al. / Food Research International 51 (2013) 954–970

with βLG in solution. The order of binding increases as the Walnut kernels have a significant amount of phenolic compounds,
number of OH group increased with EGCG> ECG>EC>C. Molecular which are generally present in the hull. When kernels are whole-
modeling showed the participation of several amino acid residues in ground and the oil is extracted, most phenolics remain in the flour
polyphenol–protein complexation with extended H-bonding network. where they can precipitate proteins through different mechanisms,
The βLG conformation was changed in the presence of polyphenols such as hydrophobic and ionic interactions, hydrogen and covalent
with an increase in β-sheet and α-helix, suggesting stronger structural bonds. It has been reported that phenolic compounds obtained from
stabilization of the protein (Kanakis et al., 2011). the whole kernels decreased protein solubility. This was affected
In the study of Wu et al. (2011) binding interaction between EGC strongly by the solvent system. Proteins from whole kernels, especial-
and βLG was investigated using fluorescence spectra, CD spectra, ly those extracted with water and NaCl solution, reduced the protein
infrared spectroscopy, and synchronous fluorescence spectra. The solubility, suggesting that phenolic compounds bind to proteins when
changes in negative entropy and the enthalpy indicated that the they are dispersed in aqueous media at neutral pH (Relkin & Shukat,
interaction between EGC and βLG was driven mainly by van der 2012). The study of Van Koningsveld et al. (2002) also confirmed that
Waals interactions and hydrogen bonding. This also indicated that phenolic compounds may be responsible for the low solubility of
the surface of βLG was covered by EGC, resulting in change of native some potato protein preparations.
conformation of βLG (Wu et al., 2011). The interactions of proanthocyanidins with proteins can also mod-
Roy et al. (2012) also investigated the interactions of two stereo- ify the functional properties of food proteins, as these interactions
isomeric antioxidant flavonoids, C and EC with BSA and human often result in a decrease of protein solubility. In apple juice, in
serum albumin (HSA) by steady state and time resolved fluorescence, which the main phenolic compound is the dimeric procyanidin B2,
phosphorescence, CD, FTIR and protein–ligand docking studies. The it was observed that a higher ionic strength decreases protein solubil-
steady-state fluorescence studies indicated a single binding site for ity after several days of incubation (Tajchakavit, Boye, Bélanger, &
both the ligands. FTIR spectra suggest that in both the albumins, C Couture, 2001).
and EC stabilize the α-helix at the cost of a corresponding loss in In the study of Rawel et al. (2002) soy glycinin and soy trypsin inhib-
the β-sheet structure. CD studies have been carried out using (+)-C, itor were derivatized by chlorogenic and caffeic acid (cinnamic acids,
and both the epimers (+)-C and (−)-C. The low temperature phos- C6\C3 structure), and by gallic acid representing hydroxybenzoic
phorescence and protein–ligand [(+), (−) and (±) forms of C and acids (C6\C1 structure). Further, the flavonoids, flavone, apigenin,
EC] docking studies indicated that the ligands bind in the proximity kaempferol, quercetin and myricetin (C6\C3\C6 structure) were
of Trp 134 of BSA and Trp 214 of HSA, thereby changing their solvent also caused to react with soy proteins to estimate the influence of the
accessible surface areas (Roy et al., 2012). number and the position of hydroxy substituents. The derivatives
It was observed that the protein–phenolic interactions increase were characterized in terms of their solubility at different pH values to
the molecular weight of proteins. In a study of Prigent et al. (2003) document the influence on the functional properties.
molecular weight of α-lactalbumin and lysozyme was observed to It was observed that the reaction of phenolic compounds with
be increased after incubating with CGA at pH 7.0 from 680 to proteins may induce cross-linking of the proteins. These interactions
690 Da. It was suggested that this may be resulted from covalent in- also change the net charge in the protein molecules, which in turn af-
teractions between proteins and quinones formed by heat oxidation fects the solubility of the derivatives. The secondary and tertiary
of phenolic compounds (Prigent et al., 2003). Rawel et al. (2002) structures of the proteins change as a result of these interactions,
also studied the interactions between the soy proteins and phenolics influencing the surface properties of the molecules making them
such as CGA, caffeic acid and gallic acid. They have found out that hydrophilic in nature. This change in hydrophilic/hydrophobic prop-
these interactions caused the formation of high molecular weight erties may affect not only the solubility behavior, but also other func-
fractions as well. tional properties like emulsification, foaming properties and gelation
of the derivatives (Rawel et al., 2002).
3.2. Effects on functional properties On the other hand, interaction of proteins with phenolic com-
pounds may improve the thermal stability of proteins. Tsai and She
Proteins have several functional properties in food systems such (2006) observed that the thermal stability of SOD was increased
as solubility, water absorption and binding, modifying viscosity, after its interaction with phenolic compounds. Application of higher
gelation, adhesion, elasticity, plasticity, emulsification, fat absorption, temperature resulted in higher binding capacity of phenolic compounds
color and flavor binding, foaming, catalysis, and fiber formation. to proteins. However, heating also led to the disruption of the protein–
Depending on the food systems and the proteins involved, such phenolic complex. It has been reported that SOD activity was higher
functions may be desirable (such as the use of egg proteins as a after incubating with phenolic compounds, and hydroxycinnamic acids
foaming agent) or undesirable (such as enzymatic browning of fruits had more effect than hydroxybenzoic acid on SOD activity; this was due
and vegetables) for foods. The distinctive functional properties of to the resonance structure of hydroxycinnamic acids having the capability
various proteins make them crucial for the production of some to support the stability of SOD through incubation. The antioxidant capac-
foods (e.g., wheat gluten is a unique protein for the elasticity and ity of peas increased after interacting with phenolic compounds and
plasticity of the dough) (Sathe, 2012). coumaric acid provided the maximum antioxidant activity to peas
Protein solubility or insolubility is an important factor for under- compared to gallic acid, catechin, ferulic acid and caffeic acid (Tsai &
standing the performance of functionality of the protein in food She, 2006). The increase in antioxidant activity in peas was as a result of
systems since protein insolubility may also limit other functional protein–phenolic interactions in peas which stabilized the protein, gener-
properties of proteins. Protein solubility depends on some of the ally SOD, and provided the antioxidant capacity for the protein during
intrinsic (e.g., protein amino acid composition, protein amino acid heating (Tsai & She, 2006). It was as a result of the stronger binding of
sequence) and extrinsic (pH, temperature, ionic strength) factors CGA with the native BSA than the denatured BSA. It was also found
(Sathe, 2012). The presence of phenolic compounds also affects pro- that there was a slight decrease in the denaturation temperature
tein solubility. Prigent et al. (2003) reported that there was a decrease when lysozyme interacts with CGA. This decrease occurred as a
in the solubility of lysozyme in the presence of CGA at pH ≥ 8.0 result of the stronger binding effect between CGA and unfolded ly-
according to the oxidation of CGA to form quinones into basic solu- sozyme. Another reason was the enhanced destabilization and
tion. Similar reduction in lysozyme and myoglobin was also observed unfolding of lysozyme. Besides, the denaturation temperature and
after the reaction of proteins and phenolic compounds (Kroll, Rawel, denaturation enthalpy of α-lactalbumin and the denaturation en-
Rohn, & Czajka, 2001). thalpy of lysozyme were not affected by CGA (Prigent et al., 2003).
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 961

A transfer in isoelectric points of soy protein to more acidic pH 4. Effects of protein–phenolic compounds interactions on
values was observed after incubating with different kinds of pheno- phenolic compounds
lic compounds (Kroll et al., 2001; Rawel et al., 2002).
In another study, the stabilization of type I collagen was investi- The antimicrobial activity of polyphenols has been extensively in-
gated using the plant polyphenol catechin. These results showed a vestigated against a wide rande of microorganism. Among polyphe-
shrinkage at 70 °C implying that catechin was able to impart thermal nols, flavan-3-ols, flavonols and tannins received most attention due
stability to collagen (Madhan, Subramanian, Rao, Nair, & Ramasami, to their wide spectrum and higher antimicrobial activity in compari-
2005). son with other polyphenols. They are able to suppress a number
of microbial virulence factors (such as inhibition of biofilm formation,
reduction of host ligands adhesion, and neutralization of bacterial
3.3. Effects on nutritional value and digestibility toxins) and show synergism with antibiotics (Daglia, 2012). Protein-
polyphenol interactions may decrease the antimicrobial capacities of
A better understanding of the factors that influence the nutritional polyphenols. Von Staszewski, Pilosof, and Jagus (2011) evaluated the
value of proteins is necessary to optimize the biological utilization of changes of antimicrobial capacities of different Argentinean green tea
proteins for human and animal nutrition. Nutritional characteristics varieties by addition of whey proteins. The results revealed some de-
of proteins after their interaction with phenolic compounds should gree of masking in the antimicrobial activity of green tea infusions
be evaluated. when whey proteins are added. The antimicrobial effects in the pres-
Polyphenols interact with salivary proteins, especially with proline- ence of whey proteins correlated with the polyphenol content of the
rich proteins (PRPs), forming insoluble aggregates that are supposed to green tea infusions and increased with the reduction of whey protein
be at the origin of astringency sensation (Soares et al., 2011). Soares, concentration. The antimicrobial effect and potential was similar within
Mateus, and Fernandes (2012) studied procyanidin trimer and PGG a pH range from 4.0 to 7.0, allowing its application to a wide group of
(pentagalloylglucose) interactions, solubility (both by HPLC) and size foods (Von Staszewski et al., 2011). There are several other studies deal-
of the complexes formed by dynamic light scattering. The results ing with the changes that occur as a result of protein–phenolic interac-
showed that mainly acidic PRPs (aPRPs) and statherin interact with tions. However, in this review mainly effects on antioxidants and
tannins, forming a significant quantity of complexes (either insoluble bioavailability of phenolic compounds are evaluated.
or soluble), while bPRPs (basic PRPs) interact poorly with procyanidin
trimer and gPRPs (glycosylated PRPs) only complex with PGG. In gener-
al, PGG formed a high amount of insoluble complex with salivary pro- 4.1. Effects on total phenolic content (TPC), total flavonoid content (TFC)
teins while procyanidin trimer formed soluble complexes (except for and antioxidant activity
statherin). These results highlighted the influence and mechanisms of
different tannins and salivary proteins that could present in their inter- The measurement of the antioxidant capacity of food products is a
action, and consequently in the development of the astringency sensa- matter of growing interest because it may provide a variety of infor-
tion (Soares et al., 2012). mation, such as resistance to oxidation, quantitative contribution of
Rawel et al. (2002) reported reductions in the amounts of lysine, antioxidant substances, or the antioxidant activity that they may
cysteine and tryptophan determined in soy proteins after interacting present inside the organism when ingested (Huang, Ou, & Prior,
with different phenolic compounds. Soy glycinin and soy trypsin inhib- 2005; Serrano, Goñi, & Saura-Calixto, 2007). Numerous in vitro stud-
itor were derivatized by chlorogenic and caffeic acids (cinnamic acids, ies have been conducted to evaluate the total antioxidant capacity
C6\C3 structure), and by gallic acid representing hydroxybenzoic (TAC) of food products. So far, however, there is no official standard-
acids (C6\C1 structure). Further, the flavonoids, flavone, apigenin, ized method, and therefore it is recommended that each evaluation
kaempferol, quercetin and myricetin (C6\C3\C6 structure) were should be performed under different oxidation conditions and differ-
also reacted with soy proteins. They have estimated the influence of ent measurement methods. The methods for measuring antioxidant
the number and the position of hydroxy substituents of these flavonoids capacity are basically classified into two groups, depending on the re-
on selected physicochemical properties of the soy proteins. The deri- action mechanism: methods based on hydrogen atom transfer and
vation caused a reduction of lysine, cysteine and tryptophan residues methods based on electron transfer (Huang et al., 2005). The majority
in soy proteins. These results underlined the possible nutritional conse- of hydrogen atom transfer-based assays apply a competitive scheme,
quence of protein–phenolic interactions by affecting the bioavailability in which antioxidant and substrate compete for thermally generated
of the essential amino acids in the food systems (Rawel et al., 2002). peroxyl radicals through the decomposition of azo-compounds. Elec-
It has been also reported that the presence of condensed tannins tron transfer based assays measure the capacity of an antioxidant in
decreased in vivo and in vitro digestibility of proteins. Their interac- the reduction of an oxidant, which results in color changes when ox-
tions form indigestible proteins and inhibitory digestive enzymes. It idation occurs. The degree of color change is correlated with the
was observed that sorghum-condensed tannins can complex kafirin sample's antioxidant concentration (Zulueta, Esteve, & Frigola, 2009).
which is one of the main proteins of sorghum. As a result of this com- In recent years, a wide range of spectrophotometric assays has
plexation, a decrease in protein digestibility of high-tannin sorghum been adopted to measure antioxidant capacity of foods, the most pop-
was observed (Emmambux & Taylor, 2003). Duodu et al. (2003) ular ones are 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid
also reviewed the factors affecting the sorghum digestibility and clas- (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, among
sified the phenolic compounds depending on the exogenous factors others such as oxygen radical absorbance capacity (ORAC), copper re-
affecting protein digestibility. ducing antioxidant capacity (CUPRAC) and ferric reducing antioxi-
In another study, leaves harvested from Acacia drepanolobium, dant power (FRAP) assays. These antioxidant capacity measurement
Acacia nilotica, Acacia seyal, Acacia tortilis, Acacia polyacantha and assays are used widely for all kinds of foods such as vegetables and
Acacia senegal were studied and it was observed that tannins have fruits, cocoa and cocoa products, beverages including fruit juices, tea
negative influence on digestibility of proteins in vitro (Rubanza et and coffee etc. There are many studies on the effect of proteins on
al., 2005). Similarly, another study, on the effect of sea buckthorn the antioxidant capacity of phenolic compounds influenced by the
procyanidins on the protein digestibility also demonstrated that protein–phenolic compound interactions. A brief outline on the effects
sea buckthorn procyanidins precipitated proteins and inhibited di- of protein–phenolic compound interactions on the total phenolic and
gestive enzymes which may have changed the digestion of proteins flavonoid contents and total antioxidant capacities of polyphenol rich
(Arimboor & Arumughan, 2011). food products including the methods of analysis are given in Table 2.
962 T. Ozdal et al. / Food Research International 51 (2013) 954–970

Almajano et al. (2007) mixed BSA, β-lactoglobulin, α-lactalbumin, They reported that the lowest total phenolic content, total flavonoid
β-casein and α-casein with epigallocatechin gallate (EGCG) at 30 °C, content and antioxidant capacities were observed in milk chocolate
resulted with the formation of an adduct having antioxidant activity. although it contains higher cocoa solids content (29%) than cocoa bars
The antioxidant activity of the protein component, which included (16%). It was suggested that this decrease was as a result of strong
both unmodified protein and the protein–EGCG adduct, increased catechin–protein interactions. Milk based products represent a very
with storage time at 30 °C using ABTS, FRAP and ORAC methods complex matrix where strong catechin–protein interactions are well-
(Almajano et al., 2007). known to occur and it directly influences catechin determination by sig-
Belščak et al. (2009) studied the total phenolic contents, total flavo- nificantly reducing analytical recovery from the food matrix (Belščak
noid contents and antioxidant capacities of various chocolate products. et al., 2009).

Table 2
Effect of protein–phenolic interactions on total phenolics, flavonoids and antioxidant capacity.

Product Proteins Phenolics Antioxidant Total Results Reference

capacity phenolic
measurement content
methods assays

Milk proteins and Bovine serum Epigallocatechin gallate (EGCG) ABTS (+) TAC Almajano et al.
BSA & EGCG albumin (BSA) FRAP (+) TAC (2007)
α-lactalbumin, ORAC (+) TAC
β-lactoglobulin,
α-casein,
β-casein.
Dairy products & Milk proteins Cocoa liquor, cocoa powder, TP (−) TP Belščak, Komes,
confectionary chocolate with (88, 72, 60%) cocoa Milk chocolate (29% cs) Horžić, Ganić, and
products solids (cs), cooking chocolate, b cocoa bar (16%cs) Karlović (2009)
powdered chocolate, milk chocolate TF (−) TF
(29% cs), cocoa bar (16% cs) Milk chocolate (29% cs)
b cocoa bar (16%cs)
ABTS (−) TAC
Milk chocolate (29% cs)
b cocoa bar (16%cs)
DPPH (−) TAC
Milk chocolate (29% cs)
b cocoa bar (16%cs)
FRAP (−) TAC
Milk chocolate (29% cs)
b cocoa bar (16%cs)
Dairy products & tea Milk proteins Tea polyphenols (Green tea, TP (−) TP Dubeau et al. (2010)
(2% skim milk) Darjeeling tea, English breakfast ABTS (−) TAC (6.0%, 8.3%, and 19.6%)
tea respectively) Voltammetry (−) TAC 4 times larger results
than ABTS
Lipid (+) TAC 19%, 10% and 12%
peroxidation
Dairy products & tea Milk proteins Tea polyphenols TP (−) TP Sharma, Vijaykumar,
DPPH (−) TAC and Jaganmohanrao
(sugar + milk) Black tea > black (2008)
tea + sugar >black
tea + milk > black
tea + milk + sugar
β-carotene-linoleic-acid assay
(+) TAC
Dairy products & tea Milk proteins Tea polyphenols (Green and ABTS () TAC Arts et al. (2002)
black tea)
Walnut kernels Walnut proteins Walnut phenolics TP Whole kernel b hull Labuckas et al.
(mostly glutelin DPPH Whole kernel b hull (2008)
and globulin)
Dairy products & tea Milk Tea phenolics FRAP (−) TAC Ryan and Petit
(semi-skimmed (2010)
and skimmed
milk)
Dairy products/soya Semi-skimmed Black tea phenolics FRAP (−) TAC (semi-skimmed Ryan and Sutherland
milk & tea bovine milk bovine milk + tea) (2011)
Soya milk 0/(+) TAC (soya milk + black tea)
Whey protein & tea Whey protein Argentinean black tea TP (−) TP Von Staszewski et al.
(8% or 16% w/v) (2011)
Coffee & milk Milk proteins Espresso, Turkish, Instant TP (−) TP Niseteo, Komes,
and Filter Coffee TF (−) TF Belščak-Cvitanović,
ABTS (−) TAC Horžić, and Budeč
FRAP (−) TAC (2012)
Coffee & milk Milk proteins Espresso TP (−) TP Sanchez-Gonzalez,
ABTS (−) TAC Jimenez-Escrig, and
FRAP (−) TAC Saura-Calixto (2005)
Coffee & tea & milk Milk proteins Tea and coffee Coloumetric (−) TAC Ziyatdinova,
FRAP method Nizamova, and
Budnikov (2010)

(−): decrease, (+): increase, TAC: total antioxidant capacity, TP: total phenolics, TF: total flavonoids.
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 963

Studies investigating the effect of milk on antioxidant capacities of effects of milk addition. The results revealed that in comparison to
tea samples have presented contradictory results, possibly because of plain brews prepared only with water, the addition of milk decreased
the different methods used for the measurement of antioxidant ca- the TPC and TFC of coffee and decaffeinated coffee brews, as opposed
pacity. Dubeau et al. (2010) measured the antioxidant capacities by to instant cappuccino brews prepared with milk, which exhibit higher
using three complementary assays; ABTS free radical scavenging, TPC when compared to plain water-made brews. Milk containing food
voltammetry, and lipid peroxidation inhibition. They observed that products represent a very complex matrix where strong polyphenol–
according to ABTS and voltammetry methods, milk decreased the anti- protein interactions are well known to occur (Siebert, Troukhanover,
oxidant capacities of teas. In contrast, in the lipid peroxidation method, & Lynn, 1996) and can directly interfere with accurate polyphenol de-
the results indicated that milk enhanced the chain-breaking antioxidant termination by significantly reducing analytical recovery, which may
capacity of teas. The researchers explained these findings as milk can be the reason for the overestimation of milk-prepared cappuccino
have dual effects on the antioxidant capacity of tea; an inhibitory effect brews. Contrary to the results obtained for coffee brews prepared
for reactions taking place in solution or at a solid–liquid interface and an with milk, instant cappuccino brews made with milk exhibited higher
enhancing effect for those in oil-in-water emulsions (Dubeau et al., TPC and TFC than those made with water. The higher values of TPC
2010). and TFC in cappuccino brews with milk can be explained by the pres-
A similar interaction between the tea phenolic compounds and milk ence of milk derived compounds and reduced analytical accuracy, as
proteins has also been reported by Sharma et al. (2008) for black tea, well as the lack of selectivity of the Folin–Ciocalteu reagent used for
with sugar, milk or both added. They reported that black tea extracts TPC analysis which reacts not only with phenols but also with other re-
had the maximum amount of polyphenol content followed by black ducing compounds such as carotenoids, amino acids, sugars and vita-
tea with sugar and black tea with both milk and sugar. In addition, min C (Vinson, Su, Zubik, & Bose, 2001). Apart from instant coffee, the
total polyphenol contents were found to give the lowest values in composition of instant cappuccino includes milk powder, powdered
black tea with milk compared to other samples, probably due to either whey proteins, lactose, sugar, vegetable fat, salt, aroma and E99 (calci-
covalent or non-covalent interactions between plant phenolics and pro- um lactobionate), ingredients that can interfere with the determination
teins. Arts et al. (2002) also reported that pure α, β, and κ-caseins of phenolic compounds in this assay, thus contributing to the inconsis-
masked the ABTS-scavenging capacities of green and black tea extracts tency of the results. The antioxidant capacities of coffee brews obtained
and of some pure flavonoids typically found in teas to different extents by both ABTS and FRAP assays were in accordance with TPC and TFC
probably because of covalent or non-covalent interactions. Both inter- values and as expected, decreased with the addition of milk. This can
actions might lead to precipitation of proteins through multisite inter- be explained by the fact that up to 1/3 of the quantity of CGA, as the
actions and multidentate interactions. In multisite interactions, several main antioxidant compound, interacts with milk proteins in coffee
phenolics bind to one protein molecule and in multidentate interactions (Dupas, Marsset-Baglieri, Ordonaud, Ducept, & Maillard, 2006).
one phenolic binds to several protein sites or protein molecules. These Another study on coffee brews (Sanchez-Gonzalez et al., 2005) was
interactions may cause masking of polyphenol contents on the proteins also in agreement with the results of Niseteo et al. (2012) The re-
(Arts et al., 2002). searchers studied the effect of milk on the total phenolic content and an-
Labuckas et al. (2008) studied the interactions of phenolics of wal- tioxidant capacity of espresso samples by ABTS and DPPH assays. They
nut kernel proteins (mostly glutelins and globulins) and their effect observed that when milk was added to the coffee, antioxidant activity
on total phenolic content and total antioxidant capacity using the decreased depending on increasing amount of milk (Sanchez-Gonzalez
DPPH (2,2-diphenyl-1-picrylhydrazyl) method. They reported that et al., 2005).
the whole kernel has a lower antioxidant capacity and total phenolic Ziyatdinova et al. (2010) developed a new approach for the evalu-
content compared to the hull. This is because the hull contains most ation of total free polyphenols content in the presence of proteins
of the phenolics and, therefore, their interactions with proteins may using electrogenerated [Fe(CN)6] 3− ions as coulometric titrant. Mix-
have decreased the total antioxidant capacity in the whole kernel tures of tea and coffee were prepared for further evaluating the effect
(Labuckas et al., 2008). of milk proteins on the antioxidant properties of beverages. Masking
Ryan and Petit (2010) reported that the additions of milk to tea effect of milk proteins on ferric reducing power of tea and coffee
significantly decreased the total antioxidant capacity depending on reflecting the content of free polyphenols in the system was observed.
the amount of milk compared to the additions of same volume of Similar masking effect of milk was also observed for the instant coffee
water as analyzed by the FRAP method. Besides, antioxidant activity (Ziyatdinova et al., 2010).
was decreased with increased amount of added milk. On the other
hand, Ryan and Sutherland (2011) studied the effect of adding soy 4.2. Effects on the content of individual phenolic compounds
milk or bovine milk on the total antioxidant capacity of five brands of
tea. It was observed that when compared to tea with semi-skimmed There are very few studies found in the literature about the effects
bovine milk, each of the five brands of analyzed tea samples had signif- of protein–phenolic interactions on the content of individual phenolic
icantly higher total antioxidant capacities or no change after the addi- compounds. These studies should be encouraged to understand the
tion of soy milk. It was suggested that the addition of soy milk to exact mechanism behind the protein–phenolic interactions.
black tea might be a useful alternative to semi-skimmed bovine milk Ferruzzi and Green (2006) studied the interactions of tea cate-
if maintaining the overall antioxidant potential of the tea infusion is de- chins with proteins. Their objective was to evaluate the effect of pep-
sired (Ryan & Sutherland, 2011). sin treatment as an enzymatic step to increase catechin recovery from
Von Staszewski et al. (2011) studied the total phenolic contents, milk and other formulations rich in protein. They combined brewed
antioxidant and antimicrobial activities of different Argentinean green tea with skimmed milk in ratios of 10% to 50% and analyzed
green tea varieties and whey protein mixtures. Their results revealed tea catechins by reversed phase High Performance Liquid Chromatog-
that when whey proteins are mixed with tea infusions, antioxidant raphy (HPLC) with photodiode array detection (PDA). The results in-
and antimicrobial activity of green tea infusions were masked. The dicated that recovery decreased with increasing milk content. The
degree of inhibition of antioxidant activity in each variety did not de- presence and ratio of milk most affected gallated catechins, specifical-
pend on the total polyphenol concentration, indicating the impor- ly epigallocatechin-gallate (EGCG) and epicatechin-gallate (ECG).
tance of the particular polyphenol composition of each variety (Von Bartolome et al. (2000) prepared mixtures of BSA and several low
Staszewski et al., 2011). molecular weight phenolics that were incubated and fractionated
Niseteo et al. (2012) studied the total phenolic and total flavonoid using G-50 Sephadex chromatography. Among the selected commer-
contents and antioxidant capacity of different coffee brews and the cial phenolic standards tested (p-coumaric acid, p-hydroxybenzoic
964 T. Ozdal et al. / Food Research International 51 (2013) 954–970

acid, protocatechuic acid, caffeic acid and (+)-catechin), the strongest volunteers who consumed either 200 g of blueberries plus 200 ml
BSA-binding affinity was demonstrated by 3,4-dihydroxy benzoic and of water or 200 g of blueberries plus 200 ml of whole milk. The re-
cinnamic acids (protocatechuic acid and caffeic acid), whereas sults showed that ingestion of blueberries increased plasma levels
p-hydroxybenzoic acid did not interact with BSA. Moreover, the same of reducing and chain-breaking potential and enhanced plasma con-
methodology was also applied to a phenolic extract obtained from len- centrations of caffeic and ferulic acids. However, there was no in-
tils containing p-coumaric acid, a p-coumaric acid derivative, (+)-cate- crease in plasma antioxidant capacity when blueberries and milk
chin and procyanidins B3 and B1. Interactions between lentil phenolic were ingested. There was a reduction in the peak plasma concentra-
extracts and BSA were comparable to those observed among the com- tions of caffeic and ferulic acid as well as the overall absorption of
mercial standards and BSA (Bartolome et al., 2000). caffeic acid. As a conclusion of this study, the researchers suggested
According to the results of these studies, it is obvious that different that milk and blueberry interactions impair the in vivo antioxidant
phenolic compounds might show different behaviors during their in- properties of blueberries and reduce the caffeic acid absorption
teractions with proteins. Taking into account that there are many (Serafini et al., 2009).
types of phenolic compounds present in food systems, further studies On the other hand, there are contradictory results about the effect of
on individual phenolic compounds are of critical importance to un- milk on the plasma antioxidant capacity as reviewed by Lotito and Frei
derstand their exact role and behavior. (2006). It has been suggested that polypeptide chains are located
around the fat globule membrane, which promotes their linkage with
4.3. Effects on in-vivo bioavailability phenolic compounds (Serafini et al., 2009) and probably results with a
decrease in the accessibility of the colonic microbiota and thus their
Proteins affect the antioxidant activity of polyphenols mostly in a degradation (Lotito & Frei, 2006). Recently, the same behavior was ob-
negative way. They usually decrease the antioxidant activity due to served in yogurt. Roowi et al. (2009) investigated the catabolism of
their strong binding affinity to polyphenols. However, the effect of hesperetin-7-O-rutinoside following acute supplementation of orange
protein–polyphenolic compound interactions should also be consid- juice, with and without yogurt. The orange juice was found to contain
ered from the bioavailability of phenolic compounds point of view. hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside. GC–MS
It is important to determine whether protein interaction with poly- (gas chromatography–mass spectrometry) analysis of the urine sam-
phenols modulate the uptake and concentration of polyphenols in ples resulted with the identification of nine phenolic acids, five of
plasma. A brief outline on the effects of protein–phenolic interactions which, 3-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid,
on the bioavailability of phenolics is presented in Table 3. dihydroferulic acid, 3-methoxy-4-hydroxyphenylhydracrylic acid and
Keogh et al. (2007) conducted experimental studies with human 3-hydroxyhippuric acid, were related with orange juice consumption sig-
subjects consisting of 24 middle-aged men and women evaluating nifying that they were derived from colonic catabolism of hesperetin-
whether milk proteins with cocoa polyphenols modulate the uptake 7-O-rutinoside. The overall (0–24 h) excretion of the five phenolic acids
and concentration of polyphenols in plasma. It was proved that milk increased significantly, equivalent to 37% of the ingested flavanones, fol-
powder did not influence the average concentration of polyphenols. lowing orange juice consumption. When the orange juice was ingested
Although it slightly accelerated absorption, this was of no physiolog- with yogurt, excretion fell back markedly (Roowi et al., 2009).
ical significance (Keogh et al., 2007). In another study, Green et al. (2007) characterized the effect of com-
Duarte and Farah (2011) also studied the effect of simultaneous con- mon food additives on digestive recovery of tea catechins. Green tea
sumption of coffee and milk on the urinary excretion of CGA and metab- water extracts were prepared containing EC, EGC, EGCG and ECG, re-
olites. Experimental subjects were submitted to consumption of water, spectively. Bovine, soy rice milks were added to understand the impact
instant coffee dissolved in water and instant coffee dissolved in milk. of creaming agents on catechin digestive recovery. Samples were ana-
After 24 h of consumption, the urine of the subjects was collected to ana- lyzed with in vitro digestive method which simulated gastric and
lyze CGA and metabolites by HPLC/LC–MS (liquid chromatography-mass small intestinal conditions with pre- and post- digestion catechin pro-
spectrometry). It was observed that the amount of CGA and metabolites files measured by HPLC. Tea samples enriched with 50% bovine, soy
recovered after consumption of combined coffee-milk (40%±27%) was and rice milk increased the recovery of total catechins by 52, 55 and
consistently lower in all subjects compared to that of coffee alone 69%, respectively. On the other hand, in vivo methods which represent
(68%±20%). It was suggested that the simultaneous consumption of the dynamic nature and heterogeneity of the gastrointestinal tract did
milk and coffee may impair the bioavailability of coffee CGA in humans not give any significant difference between the control sample and
(Duarte & Farah, 2011). teas enriched with bovine, rice and soy milk. According to the results,
In another study conducted in vivo, it was investigated whether the there were no significant differences found in catechin bioavailability
interaction of milk reduces the bioaccessibility of tea catechins associat- and plasma antioxidant activity between tea and tea-milk beverage in
ed with tea beneficial effects. Addition of milk to black tea resulted with humans in in vivo observations. As a result, it was concluded that the re-
the formation of polyphenol–protein complexes, and a decrease in total sults of in vitro methods cannot be fully and directly extended to in vivo
catechin recovery. Polyphenol–protein complexes were degraded dur- effects (Green et al., 2007).
ing digestion. It was very unlikely that consumption of tea with or with- Arts et al. (2001) studied the interactions between human blood
out milk will result in a difference in catechin plasma concentration. plasma and quercetin, rutin, catechin and 7-monohydroxyethylrutoside
This result is in disagreement with other studies in which a decrease using the ABTS method. They found that the antioxidant capacity of
in the polyphenol bioavailability was observed by the effect of human blood plasma enriched with these phenolic compounds was
protein–phenolic interactions (Burg-Koorevaar et al., 2011). lower than the sum of antioxidant capacities of both components. This
Kyle et al. (2007) also studied the addition of milk to tea on ab- effect was found to be much lower in deproteinated plasma. Therefore,
sorption of polyphenols. It was found that consumption of black tea it was attributed to the interactions of catechols with human blood
was associated with significant increases in plasma antioxidant capacity plasma.
and concentrations of total phenols, catechins, and the flavonols quer- Leenen et al. (2000) also studied the effect of milk addition on the
cetin and kaempferol within 80 min. However, the polyphenol absorp- plasma antioxidant capacity of black and green tea samples in vivo.
tion was unaffected by the addition of milk to black tea (Kyle et al., Blood samples were obtained at baseline and at several time points
2007). up to 2 h post-tea drinking. Plasma was analyzed for total catechins
Serafini et al. (2009) studied the bioavailability of phenolics and in and antioxidant activity, using the FRAP assay. The results indicated
vivo antioxidant capacity of blueberries consumed with and without that addition of milk to black or green tea did not significantly affect
milk by conducting the experiments with eleven healthy human the plasma antioxidant activity. In another study by Reddy et al.
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 965

Table 3
Effects of protein–phenolic interactions on bioavailability of phenolics.

Product Proteins Phenolics Methods Results Reference

In vivo methods
Dairy products Milk proteins Cocoa phenolics (catechins HPLC (Blood samples at 0, 0.5, 1, 1.5, (=) for C and EC Keogh, McInerney,
& cocoa and epicatechins) 2.3, 4, 6, 8 h, catechin (C) and (=) B and Clifton (2007)
epicatechin(EC) analysis)
Dairy products Milk proteins Coffee phenolics (CGA and HPLC/LC–MS (urine samples after (−) CGA and metabolites Duarte and Farah
& coffee metabolites) 24 h) (−) B (2011)
Dairy products Milk proteins Tea phenolics (English and gastric, small intestinal, and brush (−) TCAT Burg-Koorevaar,
& tea (skimmed or Indian tea catechins) border digestion, total catechin (−) B Miret, and
full-fat milk) (TCAT) recovery Duchateau (2011)
Dairy products Milk proteins Tea phenolics (catechins, HPLC (blood samples 10 min before (=) TCAT Kyle, Morrice,
& tea quercetin, kaempferol) the volunteer drank one of the test (=) quercetin McNeill, and
beverages, then 50, 80, and 180 min (=) kaempferol Duthie (2007)
thereafter) (=) B
FRAP
Fruits & tea Milk proteins Blueberry fruit phenolics Reversed phase (RP)-HPLC (−) TAC Serafini et al. (2009)
(−) caffeic acid
(−) ferulic acid
(−) B
Dairy products Yogurt proteins Orange juice phenolics GC-MS analysis of urine (24 h) (−) B of 5 phenolic acids Roowi, Mullen,
& fruit juices (hesperetin-7-O-rutinoside (3-hydroxyphenylacetic acid, Edwards, and
and 3-hydroxyphenylhydracrylic acid, Crozier (2009)
naringenin-7-O-rutinoside) dihydroferulic acid,
3-methoxy-4-hydroxyphenylhydracrylic
acid and 3-hydroxyhippuric acid)
Dairy products Bovine, rice and Green tea phenolics HPLC (−) TCAT, (−) B Green, Murphy,
& tea soy milk epicatechin (EC), pre- and post-digestion catechin (=) TCAT, (=) B Schulz, Watkins,
proteins (50%) epigallocatechin (EGC), profiles and Ferruzzi (2007)
epigallocatechin-gallate in vivo methods
(EGCG) and
epicatechin-gallate (ECG)
Blood plasma Human blood quercetin, rutin, (+) 7-monohydroxyethylrutoside In vivo methods, ABTS (−) TAC, (−) B
& antioxidants plasma proteins catechin,
Arts et al. (2001)
Blood plasma Human blood Antioxidants (albumin, In vivo methods, ABTS (−) TAC, (−) B Błauz et al. (2008)
& antioxidants plasma proteins ascorbic acid, GSH, GSSG,
melatonin, plasma,
quercetin, uric acid)
Dairy products Milk proteins Coffee phenolics (CGA) HPLC (plasma samples) (=) CGA, (=) B Dupas et al. (2006)
& coffee In vivo methods (=) CQA, (=) B
Dairy products Milk proteins Black and green tea In vivo methods, FRAP (=) plasma antioxidant activity Leenen, Roodenburg,
& tea phenolics Tijburg, and Wiseman
(2000)
Dairy products Milk proteins Black tea phenolics In vivo methods (=) plasma antioxidant activity Reddy, Sagar,
& tea Sreeramulu, Venu,
and Raghunath
(2005)
Dairy products Whole milk Instant coffee phenolics In vivo methods (=) caffeic acid (CA), ferulic acid (FA), Renouf et al. (2010)
& coffee or nondairy Liquid chromatography- and isoferulic acid (iFA) equivalents
creamer Electrospray ionization-tandem (=) CGA
MS analyses and quantification (=) B
of coffee phenolics in plasma (=) plasma phenolics
Dairy products Milk proteins Black and green tea HPLC (human plasma samples (=) plasma phenolics Hollman, Van Het
& tea phenolics (quercetin and every 2 h) (=) B Hof, Tijburg, and
kaempferol glycosides) In vivo methods Katan (2001)
Dairy products Milk proteins Black tea phenolics Flow-mediated dilation (FMD) (−) FMD Lorenz et al. (2007)
& tea nitro-mediated dilation (NMD) (−) favorable health effects of tea
measured by high-resolution on vascular function
vascular ultrasound, also (−) B
performed in isolated
rat aortic rings and endothelial
cells (before and 2 h after
consumption)
HPLC
Confectionary Milk proteins Chocolate phenolics Human in vivo pharmacokinetics (=) B (flavan-3-ols) Neilson et al. (2009)
& milk (flavan-3-ols) RP-HPLC
Dairy products Milk proteins Cocoa powder flavonoids LC–MS/MS (urine samples (0–6, Phenolic acids Urpi-Sarda et al.
& cocoa 6–12, and 12–24 h)). (−) 3,4-dihydroxyphenylacetic, (2010)
protocatechuic, 4-hydroxybenzoic,
4-hydroxyhippuric, hippuric, caffeic,
and ferulic acids
(+) vanillic and phenylacetic acids
Dairy products Milk proteins Cocoa powder flavonoids LC–MS/MS (plasma samples after (=) (−)-EC Roura et al. (2007)
& cocoa 2 h of intake)
Dairy products Milk proteins Chocolate flavonoids In vivo methods (−)(−)-EC Serafini et al. (2003)
& chocolate FRAP

(continued on next page)

966 T. Ozdal et al. / Food Research International 51 (2013) 954–970

Table 3 (continued)
Product Proteins Phenolics Methods Results Reference

In vivo methods
Dairy products Milk proteins Cocoa beverage flavonoids In vivo methods (=) (−)-EC Schroeter, Holt,
& cocoa HPLC Orozco, Schmitz,
and Keen (2003)
Dairy products Milk proteins Cocoa flavonols In vivo methods (=) flavonols Schramm et al.
& cocoa HPLC (2003)

In vitro methods
Dairy products Milk proteins Coffee phenolics (CGA) Caco-2 cell model coupled with an in (=) CGA, (=) B Dupas et al. (2006)
& coffee vitro digestion process (=) CQA, (=) B
Fruits & bovine Bovine serum Sea buckthorn In vitro digestion (−) B Arimboor and
serum albumin proanthocyanidins Arumughan (2011)
albumin
Confectionary Milk proteins Chocolate phenolics In vitro Digestion (=) B (flavan-3-ols) Neilson et al. (2009)
& milk (flavan-3-ols) Caco-2-cell culture experiments
Dairy products Milk proteins Black and green tea Colourimetric method by using (−) B Nizamova, Ziyatdinova,
& tea (casein and BSA) phenolics (quercetin, electrogenated bromine and Budnikov (2011)
rutin)

(=): no significant difference, (+): increase, (−): decrease, B: bioavailability of phenolics, TCAT: total catechins, TAC: total antioxidant capacity.

(2005), the findings were in agreement with the results of Leenen et were analyzed by LC–MS/MS after solid-phase extraction. Of the 15 me-
al. (2000). They investigated the effect of milk addition to black tea on tabolites assessed, the excretion of 9 phenolic acids was affected by the in-
the ability to modulate oxidative stress and antioxidant status in take of milk. The urinary concentration of 3,4-dihydroxyphenylacetic,
adult male human volunteers. It did not affect the beneficial effects protocatechuic, 4-hydroxybenzoic, 4-hydroxyhippuric, hippuric, caffeic,
of black tea on total plasma antioxidant activity, and plasma resis- and ferulic acids diminished after the intake of cocoa with milk, whereas
tance to oxidation induced ex vivo. It decreased plasma and urinary urinary concentrations of vanillic and phenylacetic acids increased. In
thiobarbituric acid reactive substance levels. The results suggested conclusion, milk moderately affected the formation of microbial phenolic
that the addition of milk may not obviate the ability of black tea to acids derived from the colonic degradation of procyanidins and other
modulate the antioxidant status of subjects and that consumption of compounds present in cocoa powder (Urpi-Sarda et al., 2010).
black tea with/without milk prevents oxidative damage in vivo Roura et al. (2007) studied the effect of milk on the absorption of
(Reddy et al., 2005). (−)-epicatechin ((−)-EC) from cocoa powder in healthy humans.
In another study, eighteen healthy volunteers consumed two out Quantification of (−)-EC in plasma was determined by LC–MS/MS
of four supplements for three days: black tea, black tea with milk, analysis after a solid-phase extraction procedure. Milk solution, as
green tea, and water. A cup of the supplement was consumed every one of the most common ways of cocoa powder consumption,
2 h each day for a total of 8 cups per day. The supplements provided seems to have a negative effect on the absorption of polyphenols
about 100 μmol quercetin glycosides and about 60–70 micromol but it was not statistically significant (Roura et al., 2007).
kaempferol glycosides. The addition of milk to black tea did not affect There are many other studies on the bioavailability of polyphenols
the plasma concentration of quercetin or kaempferol. As a result, it from cocoa that have yielded contradictory results. Serafini et al.
was concluded that flavonols were absorbed from tea but their bio- (2003) suggested that interaction between milk proteins and choco-
availability was not affected by the addition of milk (Hollman et al., late flavonoids inhibits the absorption of (−)-epicatechin ((−)-EC)
2001). into the bloodstream. On the other hand, Schroeter et al. (2003)
Renouf et al. (2010) also studied the effect of adding whole milk or showed that there was no significant difference in the (−)-EC con-
sugar and creamer to coffee on the bioavailability of coffee phenolics. centration in the plasma after the consumption of a milk-containing
According to the results, in comparison to regular black instant coffee, or water-based cocoa beverage under isocaloric and isolipidemic
the addition of milk did not significantly alter the maximum plasma conditions. Schramm et al. (2003), after evaluating the effect of sever-
concentration (Cmax), or the time necessary to reach Cmax (tmax). al foods including sugar, milk, bread, steak, and grapefruit on the ab-
The Cmax of caffeic acid and isoferulic acid was significantly lower sorption and pharmacokinetics of cocoa flavanols, also concluded that
and the tmax of ferulic acid and isoferulic acid was significantly longer milk had no effects on flavanol absorption.
for the sugar/nondairy creamer group than for the instant coffee
group. In conclusion, adding whole milk did not change the overall 4.4. Effects on in-vitro bioavailability
bioavailability of coffee phenolic acids, whereas sugar and nondairy
creamer affected the tmax and Cmax values but not the appearance of There are several studies which investigated the effect of protein–
coffee phenolics in the plasma (Renouf et al., 2010). phenolic compound interactions on in vitro bioavailability. Dupas et
Neilson et al. (2009) studied the effect of milk addition to choco- al. (2006) studied the effect of milk addition to coffee on the bioavail-
late on the bioavailability of EC of chocolate. EC bioavailability was ability of coffee phenolics, mainly CGA. Interactions between CGA and
assessed from chocolate confections [reference dark chocolate milk proteins were investigated using an ultrafiltration technique.
(CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa bev- These interactions proved to be slightly disrupted during an in vitro
erages [sucrose milk protein (BSMP), non-nutritive sweetener milk digestion process. CGA absorption and bioavailability were then stud-
protein (BNMP)], in humans. These results suggested that the pres- ied in vitro using a Caco-2 cell model coupled with an in vitro diges-
ence of milk solids did not significantly change the bioavailability of tion process. The results revealed that CGA absorption under its
EC among chocolate confection products (Neilson et al., 2009). native form is weak, and unmodified by the addition of milk proteins,
Urpi-Sarda et al. (2010) studied the effect of milk on the bioavailability but slightly reduced by the addition of Maillard reaction products.
of cocoa flavonoids considering phase II metabolites of epicatechin. They These data showed the presence of interactions between coffee phe-
studied the effect of milk at the colonic microbial metabolism level of nolics and milk proteins, but there was no significant effect observed
the non-absorbed flavanol fraction that reaches the colon and is metabo- on the bioavailability of CGA (Dupas et al., 2006). On the other hand,
lized by the colonic microbiota into various phenolic acids. Phenolic acids Neilson et al. (2009) studied the effect of milk addition to chocolate
 T. Ozdal et al. / Food Research International 51 (2013) 954–970 967

on the bioavailability of EC of chocolate. According to the results, in that influence the protein–phenolic interactions such as salt concentra-
vitro bioaccessibility and Caco-2 accumulation did not differ between tion and addition of certain reagents. However, the number of studies
treatments. Bioavailability as measured by the areas under the serum investigating the effect of all these parameters is very limited and
concentration–time curve (AUC) was not significantly different should be further studied in more detail to optimize the process condi-
between confections which is in agreement with the studies tions and to improve the beneficial health effects of phenolics. Process
suggesting that the presence of milk does not negatively affect the conditions and products can then be designed in a way that will provide
bioavailability of cocoa flavan-3-ols (Neilson et al., 2009). the maximum beneficial health effects to the consumers, and also opti-
Arimboor and Arumughan (2011) studied the effect of sea buck- mum nutritional and functional properties can be supplied to the
thorn proanthocyanidins on in vitro digestion of proteins. It was products.
found that interactions of sea buckthorn proanthocyanidins with Protein–phenolic interactions influence the structure, functional
food proteins and digestive enzymes might alter the protein digest- and nutritional properties, and digestibility of proteins. It was ob-
ibility and phenolic bioavailability, negatively. In another study, served that the protein–phenolic interactions increase the molecular
Nizamova et al. (2011) proposed a new method for the estimation weight of proteins. The presence of phenolic compounds also affects
of the bioavailability of polyphenols using electrogenerated bromine protein solubility which is an important factor in protein functionali-
as a coulometric titrant. The titration of model solutions of casein ty, because protein insolubility also hinders other protein functional
and BSA showed that casein did not interact with electrogenerated properties. The increase in the content of phenolic compounds de-
bromine, while BSA reacted with the titrant in the ratio of 1:63. The creases the solubility of proteins. In addition, interaction of proteins
proteins bound rutin and quercetin at a high rate and thus reduced with phenolic compounds may improve the thermal stability of pro-
the bioavailability of polyphenols. The concentration of free polyphe- teins. Moreover, proteins may also lead to some undesirable changes
nol was reduced with an increase in the concentration of protein in in color and taste when they interact with phenolic compounds. Fur-
the mixture. The total antioxidant capacity of tea samples was also thermore, looking from the nutritional value and digestibility point of
determined, and it was reported that green tea possess higher total view, it was observed that protein–phenolic interactions decrease the
antioxidant capacity than the black one because of the partial oxida- nutritional value of some essential food components and also in vivo
tion of polyphenols to respective thearubigins and theaflavins at the and in vitro digestibility of proteins.
fermentation step in the production of green tea. According to the re- On the other hand, some other studies focused on the changes in
sults, the total antioxidant capacity of tea decreased with the amount phenolic compounds as a result of protein–phenolic compound interac-
of milk. Milk proteins bound tea polyphenols into complexes as a re- tions and investigations on the changes in antioxidant capacities and
sult of intermolecular interactions and reduced their bioavailability, bioavailability of phenolic compounds were performed. However, re-
accordingly (Nizamova et al., 2011). search on individual phenolic compounds is lacking. On the other
hand, although the bioactivity of a molecule mainly depends on the
5. Discussion functional group/moiety in the molecule, the differences in the molecu-
lar structure were not considered in the studies performed so far. It is
As a result of protein–phenolic interactions changes occur both on important to understand the involvement of the functional groups or
the proteins and phenolic compounds. The main changes observed in the position of these functional groups in protein–phenolic complex,
proteins are basically related with the structure, functional and nutri- and it should be investigated in detail to better understand the mecha-
tional properties, and digestibility of proteins. On the other hand, nisms taking place.
these interactions result with changes in total phenolic or flavonoid Another important drawback of the protein–phenolic interaction
contents, antioxidant activities, contents of individual phenolic com- studies is that they are generally performed using only tea, coffee,
pounds, as well as in-vitro and in-vivo bioavailability of phenolic and chocolate as phenolic compound sources and milk as the protein
compounds. Conditions such as temperature, pH, types of proteins, source. Tea and coffee are the most widely consumed beverages in
protein concentration, types and structures of phenolic compounds the world with a high antioxidant capacity and they are often con-
and several other factors affect protein–phenolic interactions. There sumed together with milk. Therefore, it is important to understand
are some contradictory results about the effect of temperature on the effect of milk proteins on the antioxidant capacity and bioavail-
the binding affinity of polyphenols to proteins. Some studies indicat- ability of coffee and tea phenolics, as they also account for most of
ed that binding affinity decreases by increasing temperature whereas the polyphenol daily intake in the countries where the fruits and veg-
some others reported vice versa. Unlike the temperature, there etables are not preferred to be eaten very often. However, it is still of
are more comparable results regarding the effects of pH on protein– critical importance to work on other food proteins such as plant-
phenolic interactions. Lower pH results with a stronger binding originated proteins, to understand the effects more clearly and to be
since the dissociation of protein leads to more binding sites at lower able to compare the effect of different protein sources on these inter-
pH values. On the other hand, the different hydrophobicity and iso- actions. On the other hand, a better understanding of the protein–
electric points of proteins, and the difference in the amino acid com- phenolic interactions for a large variety of food products may provide
position lead to differences in the binding affinity of phenolic benefit for improving the process conditions and parameters for the
compounds to some proteins. The protein concentration in the solution food products that contain both proteins and phenolic compounds.
affects the protein–phenolic interactions, thus the precipitation is de- This information can also help developing new food products with
creased after high concentrations reached in the solution. Different better nutritional quality and improved health aspects.
types and structures of phenolic compounds also affect protein–pheno- According to many of the studies reviewed in this paper, it can be
lic interactions. Some of the structural elements that influence the affin- concluded that proteins significantly decrease the antioxidant capac-
ities of polyphenols for proteins are the following (Xiao et al., 2011): (i) ity in general. There are some contradictory results between these
Methylation and methoxylation of flavonoids decreased or only slightly studies which may arise from different methodologies used to mea-
affected the affinities; (ii) hydroxylation on rings A and B of flavones sure the antioxidant capacity or total phenolic/flavonoid contents. It
and flavonols slightly enhanced the interaction and hydroxylation on is expected that a single method cannot determine all the antioxidant
the ring A of flavanones and significantly improved the affinities; (iii) compounds available in the food matrix correctly. All the methods
glycosylation of polyphenols weakened the affinities; (iv) hydrogena- have their own advantages and disadvantages. Even the results of
tion of the C2_C3 double bond of flavonoids decreased the binding the methods sharing the same principle like ABTS and DPPH can
affinities (v) galloylation of catechins and esterification of gallic acid show important differences in their response to antioxidants. Similar-
significantly improved the binding affinities. There are also other factors ly, the extraction procedure used during analysis needs special
968 T. Ozdal et al. / Food Research International 51 (2013) 954–970

attention as the stability of protein–phenolic complex might be dif- by using a homogenous population including a greater number of
ferent in different solvent systems. As mentioned above, there are subjects. Further work on this topic is still necessary to clarify the
few studies about the effect of phenolic-protein interactions on indi- controversial results obtained so far and to better understand the
vidual phenolics. Most of the studies measured the total content of mechanisms underlying protein–phenolic interactions as well as fac-
phenolics, and flavonoids or the total antioxidant capacity, but there tors affecting the degree of this interaction.
is still a need for more studies that investigate individual phenolics
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