protein phenolic interaction

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, Turkeyb Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Maslak, TR-34469, Istanbul, Turkey

a b s t r a c ta r t i c l e i n f o

Article history:Received 1 August 2012Accepted 9 February 2013

Keywords:Protein–phenolic interactionsProteinsPhenolicsTotal antioxidant capacityBioavailability

Polyphenols have become an intense focus of research interest due to their health-bene!cial effects especially in thetreatment andprevention of several chronic diseases. Polyphenols are known to formcomplexeswith proteins lead-ing to changes in the structural, functional and nutritional properties of both compounds. In this review, the effectsof protein–phenolic interactions under various conditions on protein and phenolic compound's structure and func-tionality are described. The parameters that are de!ned to affect protein–phenolic interactions are basically temper-ature, pH, protein type and concentration, and the type and structure of phenolic compounds. Even though the exactmechanismof howproteins in"uence polyphenols is still not yet known, studies on the changes in the structure andfunctional propertieswere investigated. According to these studies, secondary and tertiary structures of the proteinsare 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 alsoconcluded that proteins signi!cantly decrease the antioxidant capacity in general, but there are some controversialresults 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 lackingparts 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 conditionsshould be studied in detail to improve the food processes and providemaximumbene!cial health effects to the con-sumers with optimum nutritional and functional properties.

© 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9552. Parameters affecting interactions between protein and phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956

2.1. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9562.2. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9572.3. Types of proteins and protein concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9592.4. Types and structures of phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9592.5. Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959

3. Effects of protein–phenolic compounds interactions on proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9593.1. Effects on structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9593.2. Effects on functional properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9603.3. Effects on nutritional value and digestibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961

4. Effects of protein–phenolic compounds interactions on phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9614.1. Effects on total phenolic content (TPC), total "avonoid content (TFC) and antioxidant activity . . . . . . . . . . . . . . . . . . . . 9614.2. Effects on the content of individual phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9634.3. Effects on in-vivo bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9644.4. Effects on in-vitro bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966

5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967

Food Research International 51 (2013) 954–970

! Corresponding author at: Istanbul Technical University, Department of Food Engineering, Faculty of Chemical andMetallurgical Engineering, Maslak, TR-34469, Istanbul, Turkey. Tel.: +90212 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

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

1. Introduction

Proteins are highly complex polymers, made up of twenty differentamino acids consisting of an!-carbon atom covalently attached to a hy-drogen atom, an amino group, a carboxyl group, and a side-chain Rgroup (Fig. 1) (Damodaran, 1996). The differences in structure andfunction of proteins arise from the sequence in which the amino acidsare linked together via amide bonds. Proteins are important food com-ponents existing mostly in milk, meats (including !sh and poultry),eggs, cereals, legumes and oilseeds. They can form complexes withother food components including polyphenols leading to changes intheir structural, functional and nutritional properties. A fundamentalunderstanding of these changes as a result of interactionswith phenoliccompounds is essential from the scienti!c, industrial, and economicalpoint of view.

Phenolic compounds are chemically structured as a hydroxyl groupbonded to an aromatic ring (Fig. 2). They are secondarymetabolites, notinvolved in growth and energy metabolism in the body (Harnly,Bhagwat, & Lin, 2007). There are currently more than 8000 known phe-nolic compounds identi!ed in fruits, vegetables, seeds, and liquids(Cuykens & Claeys, 2004; Guo, Kong, & Meydani, 2009). Phenolic com-pounds can be classi!ed into two groups: basic phenolic compoundsand polyphenols (Vermerris & Nicholson, 2006). Dietary polyphenolsrepresent the main source of antioxidants for human use (Graf,Milbury, & Blumberg, 2005). The main classes of polyphenols are de-!ned according to the nature of their carbon skeleton: phenolic acids,"avonoids, and the less common stilbenes and lignans. Phenolic acidsinclude caffeic acid, ferulic acid and hydrolyzable tannins. Flavonoidscan be divided into several classes according to their degree of oxidationin the heterocycle (Guo, Kong, & Meydani, 2009). Flavonoids include"avonols (e.g., quercetin and kaempferol, themost ubiquitous"avonoidsin foods), "avones, iso"avones, "avanones, anthocyanins, "avanols(catechins-monomers and proanthocyanidin polymers, known as con-densed tannins) (Manach, Scalbert, Morand, Remesy, & Jimenez, 2004;Scalbert & Williamson, 2000).

Polyphenols have become an intense focus of research interestdue to their health-bene!cial effects especially in the treatment andprevention of cancer (Chen et al., 2011; Weng & Yen, 2012) and car-diovascular diseases (Kuriyama et al., 2006; Mursu et al., 2008). Thesuggested bene!cial 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-in"ammatory (Beara et al., 2012; Zimmer et al., 2012),antiallergenic (Chung & Champagne, 2009; Schmitz-Eiberger &Blanke, 2012), anticoagulant (Bijak et al., 2011), immune modulating(Schütz, Saß, With, Graubaum, & Grünwald, 2010), antimicrobial(Silva, Rodrigues, Feas, & Estevinho, 2012; Xia, Wu, Shi, Yang, &

Zhang, 2011), vasodilatory (Mudnic et al., 2010), and analgesic activ-ities (Santoz, Almeida, Lopez, & Souza, 2010).

Polyphenols can be oxidized by molecular oxygen with side chainamino groups of peptides at alkaline pH to quinines, leading to the for-mation of protein cross-links (Damodaran, 1996; Prodpran, Benjakul, &Phatcharat, 2012). These highly reactive quinines can irreversibly reactwith the sulfhydryl and amino groups of proteins. In addition, quininescan undergo condensation reactions, resulting in the formation of highmolecular weight brown colored pigments named as tannins. Tanninsare highly reactive and can readily combine with SH and amino groupsof proteins. Quinone–amino group reactions are known to decrease thedigestibility and bioavailability of protein-bound lysine and cysteine(Damodaran, 1996).

The phenolic group is an excellent hydrogen donor that forms hy-drogen bonds with the carboxyl group of the protein. For phenoliccompounds to have high protein af!nity, they must be small enoughto penetrate inter-!brillar regions of protein molecules, but largeenough to crosslink peptide chains at more than one point (Mulaudzi,Ndhlala, Kulkarni, & Staden, 2012). The molecular explanations of pro-tein–phenolic interaction are given in Fig. 3. The diphenol moiety of apolyphenol (Almajano, Delgado, & Gordon, 2007) is readily oxidizedto an ortoquinone, either enzymatically as in plant tissues, or by molec-ular oxygen (Damodaran, 1996; Strauss & Gibson, 2004). The quinineforms a dimer (Arimboor & Arumughan, 2011) in a side reaction, or re-acts with amino or sulfhydryl side chains of polypeptides to form cova-lent C\N or C\S bonds with the phenolic ring, with regeneration ofhydroquinone. The latter can be reoxized and bind a second polypep-tide, resulting in a cross-link (Arts, Haenen, Voss, & Bast, 2001). Other-wise, two quinines, each carrying one chain, can dimerize, producinga cross-link as well (Arts et al., 2002) (Strauss & Gibson, 2004).

The interactions of phenolic compounds with proteins may lead tochanges in physico-chemical properties of proteins such as solubility,thermal stability, and digestibility (Labuckas, Maestri, Perelló, Martínez,& Lamarque, 2008; Rawel, Kroll, & Rohn, 2001). Additionally nutritionalproperties of proteins may be affected due to the modi!cation of essen-tial amino acids and through the inhibition of proteases (Kroll, Rawel, &Rohn, 2003). On the other hand, the interactions of other compounds in-cluding 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 thecharacteristics of those compounds. Polyphenols may interact with pro-teins both reversibly and irreversibly. Some of the examples from bothinteractions in the literature are given in Table 1. In reversible interac-tions, usually non-covalent forces such as hydrogen bonding, hydropho-bic bonding and van derWaals 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 interac-tions, covalent bonds are formed between the polyphenols and proteins(Haslam, 1996). A hydrogen bond is the interaction of a hydrogen atomthat is covalently attached to an electronegative atom such as N, O or Swith another electronegative atom, which is primarily an ionic interac-tion. They are only stable as long as they are protected from water. Vander Waals interactions are intermolecular interactions affected by thesurrounding solvent. They are dipole–induced dipole and induced-dipole–induced dipole interactions between neutral atoms in proteinmolecules. When two atoms come close to each other, each atominduces a dipole in the other via polarization of the electron cloud. Theinteractions between these induced dipoles have an attractive as well

COOH

H C NH2

R Fig. 1. The structure of an amino acid.

955T. Ozdal et al. / Food Research International 51 (2013) 954–970

as repulsive component. Depending on the relative number of negativelyand positively charged residues, proteins have either a net negative or anet positive charge at neutral pH. These charged groups in proteinsare distributed on the surface of the protein molecule. Electrostaticinteractions occur between like charges (repulsive interactions) andopposite charges (attractive interactions), which are charged groups onthe surface of the protein molecule and the other molecules in themedia. Hydrogen bonding and electrostatic interactions betweenvarious polar groups are not very stable, and their stabilities depend onmaintenance of an apolar environment. Hydrophobic interactionsamong nonpolar groups are stronger (Damodaran, 1996). The numberof studies which describe the reversible interactions is more thanthe irreversible ones mainly due to the lack of suitable methods forquantitating the covalent bonds between molecules.

Trombley, Loegel, Danielson, and Hagerman (2011) have suggestedthat bioactivities and bioavailability of plant polyphenols and other cat-echin derivatives may be affected by the covalent interaction betweenpolyphenols and proteins. Reaction mechanisms and methods for char-acterizing non-covalent and covalent interactions between polyphenolsand proteins were reviewed thoroughly by Bourvellec and Renard(2012), very recently.

The interactions of phenolic compounds and proteins are known toaffect the structure of proteins, content of free polyphenols, antioxidantcapacity and bioavailability of phenolic compounds in foods. A betterunderstanding of phenolic compound–protein interactions wouldhelp to control the functional properties of proteins in food products

during processing, transportation and storage. In this review, the effectsof protein–phenolic interactions under various conditions on proteinand phenolic compounds' structure and functionality are described.

2. Parameters affecting interactions between protein andphenolic compounds

There are many parameters that affect protein–phenolic interac-tions such as temperature, pH, types of proteins, protein concentration,types and structures of phenolic compounds, salt concentration, and addi-tion of certain reagents. Complex formation of protein and phenolics re-sults from hydrogen binding and hydrophobic interactions (Hagerman& Klucher, 1986). Speci!c to protein–phenolic complex, hydrophobic in-teractions 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 formationof hydrophobic bondings, therefore, it is an important parameter in theprotein–phenolic interactions. Sastry and Rao (1990) observed that tem-perature had a signi!cant in"uence on the binding of polyphenol-free11S protein of sun"ower seed with 5-O-caffeoylquinic acid. The bindingof polyphenol-free 11S protein of sun"ower seed and 5-O-caffeoylquinicacid signi!cantly decreased as the temperature increased from 30 °C to45 °C and it completely disappeared at 55 °C. Temperature affected

Fig. 2. The structure of various phenolic compounds.

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

both the maximum amount of binding points and the binding af!nity of5-O-caffeoylquinic acid to polyphenol-free 11S protein as a result of theimportant role of hydrogen bonding in the binding of 5-O-caffeoylquinicacid by the 11S protein (Sastry & Rao, 1990). Prigent et al. (2003) studiedinteractions of bovine serumalbumin (BSA)with 5-O-caffeoylquinic acidat 5, 25 and 60 °C. They reported that the binding af!nity of 5-O-caffeoylquinic acid for BSA decreased with temperature. However,Hoffmann et al. (2006) reported that precipitation of BSA withprocyanidin derivatives was independent of temperature variations.Tsai and She (2006) evaluated the contribution of phenol–proteininteractions on the antioxidant capacity of peas after immersionwith !ve phenolics under different heating conditions between 30and 70 °C. They extracted superoxide dismutase (SOD) enzymefrom peas, formed protein–phenolic interaction complex andmeasuredSOD activity and binding capacity of this complexwith pea protein. SODactivity found in fresh or processed fruits is very weak as a result ofprotein deformation induced by heating time and temperature. Theyreported that the heat stability of SOD increased after interacting withphenolic compounds as a result of the increase in SOD activation energycaused by the binding of phenolic compounds to protein. It was ob-served that the binding effect of phenolic compound was increasedwith temperature (Tsai & She, 2006).

2.2. pH

The precipitations of complexes resulting from polyphenol–proteininteractions are pH sensitive. The lowest solubility of polyphenol–protein complexes occurred at 0.3–3.1 pH units below the isoelectric

point of the proteins (Naczk, Grant, Zadernowski, & Barre, 2006). Un-like the temperature, pH affected only the degree of binding but notthe binding af!nity for the interaction between polyphenol-free 11Sprotein of sun"ower seed and 5-O-caffeoylquinic acid. Lower pH ledto stronger binding because the dissociation of protein had morebinding sites at lower pH (Sastry & Rao, 1990). The interactions be-tween chlorogenic acid (CGA) and several proteins such as BSA, lyso-zyme, and !-lactalbumin at pH!7 produced non-covalent bondsand the amount of CGA bound by BSA (per molecule) was somewhathigher at lower pH (Prigent et al., 2003). With the increase in pH, thecovalent interaction between lysozyme and CGA was stronger due tothe formation of more radicals or quinones from the autoxidation ofCGA at higher pH. The reactive radicals and quinones subsequentlyinteracted with proteins covalently (Prigent et al., 2003).

Naczk, Oickle, Pink, and Shahidi (1996) studied the effect of pH onthe formation of crude tannin canola extract/BSA, fetuin, gelatine, andlysozyme complexes. It was suggested that the optimal pH for precipi-tation varies for different proteins and generally it is close to the isoelec-tric point of the protein. Rawel, Meidtner, and Kroll (2005) alsoobserved higher binding af!nity for ferulic acid and CGA close to the iso-electric point of BSA. Frazier, Papadopoulou, and Green (2006) failed to!nd an effect of pH on binding of (")-epicatechin to BSA. This was alsosupported by results of Papadopoulou, Green, and Frazier (2005) andCharlton et al. (2002) and suggested that electrostatic interactions arenot amajor factor in forming the (")-epicatechin/BSA complex. Indeed,increased precipitation of protein/polyphenol complexes close to theisoelectric point may be attributed to the minimum solubility of theprotein at this pH.

Fig. 3. Reactions of a phenolic acid with amino side chains of polypeptides (Strauss & Gibson, 2004).


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

Type of interaction Interactionmechanism

Protein Phenolic compound Changes in functionality ormodi!cation

Assessment method References

Reversibly Non-covalentforces

Hydrogen bonding !-lactalbuminlysozymeBSA

Procyanidins ofvarious degree ofpolymerization (DP)

Procyanidins of medium DPcan lead to an undesirabledecrease of protein solubility,but may play a positive rolein foam stability

Isothermal titrationcalorimetry

Prigent et al.,2009

Hydrogen bonding Bovine serumalbumin (BSA)

Ferulic acid (FA) Thermal stability of BSAincreases upon binding withFA.

"uorescence, circulardichroism and isothermaltitration calorimetry

Ojha, Mishra,Hassan, andChaudhury(2012)

Van der Waalsforces+Hydrogenbonding

Bovine"-lactoglobulin

(")-epigallocatechin contribute to thefunctionality of the milkproducts

"uorescence spectra, CDspectra, infrared spectroscopy,and synchronous"uorescence spectra

Wu et al.(2011)

Hydrophobicbinding

"-casein in milk Green tea "avonoids(catechins)

Number of surfacehydrophobic sites decreasedwith phenolics

Fluorometry analysis,isothermal titrationcalorimetry

Yüksel et al.(2010)

Hydrophobicbinding

Walnut proteins Walnut phenolics The presence of phenoliccompounds decreased proteinsolubility in walnut "ourobtained from whole kernels.

SDS-PAGE Labuckas etal. (2008)

Hydrogen bondingand non-polarhydrophobicinteractions

Sorghumproteins

Sorghum tannins Precipitation of proteins, andmake them insoluble andindigestible

– Duodu,Taylor,Belton, andHamaker(2003)

Hydrophobic andhydrophilicinteractions

Milk"-lactoglubulin

Tea polyphenols The structural stabilization ofprotein increased

FTIR, CD, "uorescencespectroscopic methods

Kanakis et al.(2011)

Irreversibly Covalentbonds

Soy protein Chlorogenic-, caffeic-,gallic acid, "avones,apigenine,kaempferol,quercetin andmyricetin

1) Reduction in lysine,cysteine and tryptophan, 2)The isoelectric points shiftedto lower pH, 3) Increase inmolecular weight, 4) Morehydrophilic surface on soyprotein, 5) In"uence onsolubility

Circular dichroism, differentialscanning calorimetry (DSC)

Rawel,Czajka, Rohn,and Kroll(2002)

Fish myo!brillarprotein

Caffeic acid,catechin, ferullicacid and tannic acid

1) Enhanced mechanicalproperties with phenoliccompounds, 2) In"uence onproperties and appearance ofprotein !lms

Texture pro!le analysis, colormeasurement, lighttransmission, SDS-PAGE

Prodpran etal. (2012)

Gelatin Gallic acid and rutin 1) Increased gel strength,thermal stability, 2) Decreasein swelling

Texture pro!le analysis,rheometry, DSC, swelling tests,scanning electron microscopy,X-ray diffraction, FTIR

Yan, Li, Zhao,and Yi (2011)

Non-disulphidecovalent linkages

"-lactoglobulin Sour cherryphenolics(antocyanins)

1) Allergenicity of proteindecreased, 2) Digestibilityremained

SDS-PAGE, isoelectrofocusing,immunoblotting,size-exclusion andreverse-phase chromatography,mass spectrometry, digestibility,antioxidant activity

Tantoush etal. (2011)

Cross-linking Gelatin (Type A) Phenolic acid,quercetin, rutin

1) Mechanical strength of gelsincreased, 2) Reduced swelling,3) Fewer free amino groups,4) denser polymeric networks

Free amino groups analysis,gel rigidity, swelling, dynamiclight scattering

Strauss andGibson(2004)

Milk protein Caffeic acid 1) Enhance the heat stability ofmilk2) Reduced the lysine andsulfhydryl content of heatedmilk3) No effect on rennetcoagulation time, alcoholstability, viscosity orzeta potential4) Increased casein micelle size5) Increased crosslinking of milkproteins

Heat coagulation time-pHpro!le, determination ofavailable lysine, sulfhydrylgroups, zeta potential, relativeviscosity, measurement ofcasein micelle size

O'Connelland Fox(1999)

Cross-linking Porcine plasmaprotein

Tannic acid, caffeicacid, ferulic acid

1) Tensile strength increased,2) Elongation at breakincreased, 3) water vaporpermeability of !lms increased

Mechanical properties, watervapor permeability

Nuthong,Benjakul, andProdpran(2009)


2.3. Types of proteins and protein concentration

Protein–phenolic interaction is affected by the types of proteins andthe molar ratio of phenolic/protein (Prigent et al., 2003). They can bindeither hydrophobically or hydrophilically depending on binding sites ofthe protein. The difference in binding af!nity among proteins dependson 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 af!nity of CGA to BSAwashigher than to lysozyme and !-lactalbumin. The amount of protein inthe solution affects the protein–phenolic interactions as well. If the con-centration of BSA was low, the difference of protein precipitationbetween 0.5 mg/ml of BSA and 1.0 mg/ml of BSA was not statisticallysigni!cant. On the other hand, when the concentration of BSA washigher than 1.0 mg/ml, the protein precipitation effectwas signi!cantlylower comparing to the effect obtained from higher concentrations ofBSA (Naczk et al., 1996).

2.4. Types and structures of phenolic compounds

Different types of phenolic compounds affect protein–phenolic inter-actions depending on factors such as molecular weight, methylation, hy-droxylation, glycosylation, and hydrogenation of phenolic compounds.The binding af!nity of polyphenols to proteins increases with their mo-lecular size. Larger polyphenols like those present in black tea (thea"avin,thearubigin) aremore likely to bindmilk proteins due to the fermentativeoxidation/polymerization of catechin monomers (Dubeau, Samson, &Tajmir-Riahi, 2010). Among several lowmolecular weight phenolic com-pounds including p-coumaric acid, p-hydroxybenzoic acid, cinnamic acids(protocatechuic acid and caffeic acid) and catechin, the 3,4-dihydroxybenzoic and cinnamic acids had the strongest binding af!nity for BSAwhile there was no signi!cant interaction between p-hydroxybenzoicacid and BSA (Bartolome, Estrella, & Hernandez, 2000). Although bothquercetin and quercetin 3-O-"-D-glucopyranoside are "avonoids, theirbinding af!nities with BSA were different since stronger interaction wasobserved between BSA and quercetin (Martini, Claudia, & Claudio,2008). For increasing the heat stability of SOD in peas, hydroxycinnamicacids including ferulic acid, coumaric acid and caffeic acid were found toincrease the heat stability better than hydroxybenzoic acid; coumaricacid was found to be superior for enhancing the antioxidant activity ofSOD and showed the strongest binding ability with pea protein (Tsai &She, 2006).

Xiao et al. (2011) investigated the relationship between the struc-tural properties of dietary polyphenols and their af!nities for milkproteins. Methylation and methoxylation of "avonoids weakened orlittle affected their binding af!nities for milk proteins. In general,the methylation of hydroxyl group in "avonoids decreased their bind-ing af!nities for milk proteins by 1.10–14.79 times. The af!nity ofdaidzein for milk proteins was found to be 14.79-times higher thanthat of its methylated form (formononetin). 40-methoxylation ofgalangin hardly affected the af!nity for milk proteins. Hydroxylationon the rings A and B of "avones and "avonols slightly enhanced thebinding af!nities for milk proteins. The hydroxylation on the ring Aof "avanones signi!cantly improved the af!nities for milk proteins.However, the hydroxylation on the ring C of "avones hardly in"uencedthe binding af!nities for milk proteins and the hydroxylation on ring Aof iso"avones reduced or little affected the af!nities for milk proteins.Quercetin binding af!nities for milk proteins was found to be only1.02 times higher than that of quercitrin which is the glycoside formedfrom quercetin. The af!nities (logKa) of naringenin, naringin andnarirutin for milk proteins were determined as 3.94, 3.76, and 3.64, re-spectively. It revealed that the monoglycosides of "avonoids showedstronger binding af!nities with milk proteins than their polyglycosideforms. The decreasing af!nity for milk protein after glycosylation maybe caused by the non-planar structure. After the hydroxyl group issubstituted by a glycoside, steric hindrance may take place, which

weakens the af!nity for proteins. Glycosylation of resveratrol slightlyreduced the af!nity for milk proteins. The af!nity of resveratrol formilk proteins was about 2.51 times higher than that of polydatin. Thehydrogenation of the C2_C3 double bond of "avonoids decreased thebinding af!nities for milk proteins about 7.24 to 75.86 times. The af!n-ities of apigenin andmyricetin formilk proteinswere about 75.86 timesand 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 af!nity for BSA by 2–4 orders of mag-nitude. Galloylation of catechins signi!cantly improved the binding af-!nities with milk proteins about 100–1000 times. The pyrogallol-typecatechins showed lower af!nities than catechol-type catechins. More-over, the af!nity of catechin with 2,3-trans structure for milk proteinswas found to be higher than that of the catechin with 2,3-cis structure.The esteri!cation of gallic acid signi!cantly improved the af!nity formilkproteins. The af!nities of gallic acid and its esters with !-amylase weredetermined as: methyl gallate>ethyl gallate>propyl gallate>gallicacid (Xiao et al., 2011).

2.5. Other factors

Other factors in"uencing the protein–phenolic interactions aresalt concentration and addition of certain reagents. The bindingstrength of CGA to sun"ower 11S protein was reduced with an in-crease in NaCl concentration. The concentration of NaCl lowered theamount of binding points instead of affecting the binding af!nitydue to fact that salts at high concentration can inhibit the dissociationof oligomeric proteins (Sastry & Rao, 1990). Some reagents, such asNa2SO3 (a reducing agent), even at low concentration can affect theprotein–phenolic interactions, the binding between CGA and 11S pro-tein disappeared completely in 0.01 M Na2SO3 (Sastry & Rao, 1990).

3. Effects of protein–phenolic compounds interactions on proteins

3.1. Effects on structure

The mechanism of how proteins in"uence polyphenols is still notyet known. In order to give an explanation, !rstly the changes in thestructures of the proteins should be well understood. There are sever-al researches that have been performed recently to understand themechanism by which the antioxidants in tea are affected by the addi-tion of milk.

Hasni et al. (2011) studied the interaction of !- and "-caseinswith tea polyphenols (+)-catechin (C), (+)-epicatechin (EC),(+)-epigallocatechin (EGC) and (+)-epigallocatechin gallate (EGCG)at amolecular level, using Fourier transform infrared (FTIR), UV–visible,circular dichroism (CD), "uorescence spectroscopic methods and mo-lecular modeling. It was concluded that tea polyphenols weakly bindto!-casein and "-casein through both hydrophilic and hydrophobic in-teractions. The order of binding increases as the number of OHgroup in-creased with C~EC>EGC>EGCG. "-Casein forms stronger complexeswith tea polyphenols than !-casein, due to the more hydrophobicnature of "-casein. Structural modeling of the interaction between !-and "-caseins and tea polyphenols showed that the participation ofseveral amino acid residues in polyphenol–protein complexation withextended H-bonding network. Casein conformation was changed bypolyphenol with a major reduction of !-helix and "-sheet and increaseof random coil (Hasni et al., 2011).

Kanakis et al. (2011) investigated the interaction of "-lactogolobulin("LG) with tea polyphenols (+)-C, (")-EC, (")-ECG and (")-EGCG atmolecular level, using FTIR, CD, "uorescence spectroscopic methodsand 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 analysisit was observed that polyphenols bind "LG through both hydrophilicand hydrophobic interactions. Tea polyphenols make weak bonds


with "LG in solution. The order of binding increases as thenumber of OH group increased with EGCG>ECG>EC>C. Molecularmodeling showed the participation of several amino acid residues inpolyphenol–protein complexation with extended H-bonding network.The "LG conformation was changed in the presence of polyphenolswith an increase in "-sheet and !-helix, suggesting stronger structuralstabilization of the protein (Kanakis et al., 2011).

In the study of Wu et al. (2011) binding interaction between EGCand "LG was investigated using "uorescence spectra, CD spectra,infrared spectroscopy, and synchronous "uorescence spectra. Thechanges in negative entropy and the enthalpy indicated that theinteraction between EGC and "LG was driven mainly by van derWaals interactions and hydrogen bonding. This also indicated thatthe surface of "LG was covered by EGC, resulting in change of nativeconformation of "LG (Wu et al., 2011).

Roy et al. (2012) also investigated the interactions of two stereo-isomeric antioxidant "avonoids, C and EC with BSA and humanserum albumin (HSA) by steady state and time resolved "uorescence,phosphorescence, CD, FTIR and protein–ligand docking studies. Thesteady-state "uorescence studies indicated a single binding site forboth the ligands. FTIR spectra suggest that in both the albumins, Cand EC stabilize the !-helix at the cost of a corresponding loss inthe "-sheet structure. CD studies have been carried out using (+)-C,and both the epimers (+)-C and (")-C. The low temperature phos-phorescence and protein–ligand [(+), (") and (±) forms of C andEC] docking studies indicated that the ligands bind in the proximityof Trp 134 of BSA and Trp 214 of HSA, thereby changing their solventaccessible surface areas (Roy et al., 2012).

It was observed that the protein–phenolic interactions increasethe molecular weight of proteins. In a study of Prigent et al. (2003)molecular weight of !-lactalbumin and lysozyme was observed tobe increased after incubating with CGA at pH 7.0 from 680 to690 Da. It was suggested that this may be resulted from covalent in-teractions between proteins and quinones formed by heat oxidationof phenolic compounds (Prigent et al., 2003). Rawel et al. (2002)also studied the interactions between the soy proteins and phenolicssuch as CGA, caffeic acid and gallic acid. They have found out thatthese interactions caused the formation of high molecular weightfractions as well.

3.2. Effects on functional properties

Proteins have several functional properties in food systems suchas solubility, water absorption and binding, modifying viscosity,gelation, adhesion, elasticity, plasticity, emulsi!cation, fat absorption,color and "avor binding, foaming, catalysis, and !ber formation.Depending on the food systems and the proteins involved, suchfunctions may be desirable (such as the use of egg proteins as afoaming agent) or undesirable (such as enzymatic browning of fruitsand vegetables) for foods. The distinctive functional properties ofvarious proteins make them crucial for the production of somefoods (e.g., wheat gluten is a unique protein for the elasticity andplasticity of the dough) (Sathe, 2012).

Protein solubility or insolubility is an important factor for under-standing the performance of functionality of the protein in foodsystems since protein insolubility may also limit other functionalproperties of proteins. Protein solubility depends on some of theintrinsic (e.g., protein amino acid composition, protein amino acidsequence) and extrinsic (pH, temperature, ionic strength) factors(Sathe, 2012). The presence of phenolic compounds also affects pro-tein solubility. Prigent et al. (2003) reported that there was a decreasein the solubility of lysozyme in the presence of CGA at pH#8.0according to the oxidation of CGA to form quinones into basic solu-tion. Similar reduction in lysozyme and myoglobin was also observedafter the reaction of proteins and phenolic compounds (Kroll, Rawel,Rohn, & Czajka, 2001).

Walnut kernels have a signi!cant amount of phenolic compounds,which are generally present in the hull. When kernels are whole-ground and the oil is extracted, most phenolics remain in the "ourwhere they can precipitate proteins through different mechanisms,such as hydrophobic and ionic interactions, hydrogen and covalentbonds. It has been reported that phenolic compounds obtained fromthe whole kernels decreased protein solubility. This was affectedstrongly by the solvent system. Proteins fromwhole kernels, especial-ly those extracted with water and NaCl solution, reduced the proteinsolubility, suggesting that phenolic compounds bind to proteins whenthey are dispersed in aqueous media at neutral pH (Relkin & Shukat,2012). The study of Van Koningsveld et al. (2002) also con!rmed thatphenolic compounds may be responsible for the low solubility ofsome potato protein preparations.

The interactions of proanthocyanidins with proteins can also mod-ify the functional properties of food proteins, as these interactionsoften result in a decrease of protein solubility. In apple juice, inwhich the main phenolic compound is the dimeric procyanidin B2,it was observed that a higher ionic strength decreases protein solubil-ity after several days of incubation (Tajchakavit, Boye, Bélanger, &Couture, 2001).

In the study of Rawel et al. (2002) soy glycinin and soy trypsin inhib-itor were derivatized by chlorogenic and caffeic acid (cinnamic acids,C6\C3 structure), and by gallic acid representing hydroxybenzoicacids (C6\C1 structure). Further, the "avonoids, "avone, apigenin,kaempferol, quercetin and myricetin (C6\C3\C6 structure) werealso caused to react with soy proteins to estimate the in"uence of thenumber and the position of hydroxy substituents. The derivativeswere characterized in terms of their solubility at different pH values todocument the in"uence on the functional properties.

It was observed that the reaction of phenolic compounds withproteins may induce cross-linking of the proteins. These interactionsalso change the net charge in the protein molecules, which in turn af-fects the solubility of the derivatives. The secondary and tertiarystructures of the proteins change as a result of these interactions,in"uencing the surface properties of the molecules making themhydrophilic in nature. This change in hydrophilic/hydrophobic prop-erties may affect not only the solubility behavior, but also other func-tional properties like emulsi!cation, foaming properties and gelationof the derivatives (Rawel et al., 2002).

On the other hand, interaction of proteins with phenolic com-pounds may improve the thermal stability of proteins. Tsai and She(2006) observed that the thermal stability of SOD was increasedafter its interaction with phenolic compounds. Application of highertemperature resulted in higher binding capacity of phenolic compoundsto proteins. However, heating also led to the disruption of the protein–phenolic complex. It has been reported that SOD activity was higherafter incubating with phenolic compounds, and hydroxycinnamic acidshad more effect than hydroxybenzoic acid on SOD activity; this was dueto the resonance structure of hydroxycinnamic acids having the capabilityto support the stability of SOD through incubation. The antioxidant capac-ity of peas increased after interacting with phenolic compounds andcoumaric acid provided the maximum antioxidant activity to peascompared to gallic acid, catechin, ferulic acid and caffeic acid (Tsai &She, 2006). The increase in antioxidant activity in peas was as a result ofprotein–phenolic interactions in peaswhich stabilized the protein, gener-ally SOD, and provided the antioxidant capacity for the protein duringheating (Tsai & She, 2006). It was as a result of the stronger binding ofCGA with the native BSA than the denatured BSA. It was also foundthat there was a slight decrease in the denaturation temperaturewhen lysozyme interacts with CGA. This decrease occurred as aresult of the stronger binding effect between CGA and unfolded ly-sozyme. Another reason was the enhanced destabilization andunfolding of lysozyme. Besides, the denaturation temperature anddenaturation enthalpy of !-lactalbumin and the denaturation en-thalpy of lysozyme were not affected by CGA (Prigent et al., 2003).


A transfer in isoelectric points of soy protein to more acidic pHvalues was observed after incubating with different kinds of pheno-lic compounds (Kroll et al., 2001; Rawel et al., 2002).

In another study, the stabilization of type I collagen was investi-gated using the plant polyphenol catechin. These results showed ashrinkage at 70 °C implying that catechin was able to impart thermalstability to collagen (Madhan, Subramanian, Rao, Nair, & Ramasami,2005).

3.3. Effects on nutritional value and digestibility

A better understanding of the factors that in"uence the nutritionalvalue of proteins is necessary to optimize the biological utilization ofproteins for human and animal nutrition. Nutritional characteristicsof proteins after their interaction with phenolic compounds shouldbe evaluated.

Polyphenols interact with salivary proteins, especially with proline-rich proteins (PRPs), forming insoluble aggregates that are supposed tobe 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 sizeof the complexes formed by dynamic light scattering. The resultsshowed that mainly acidic PRPs (aPRPs) and statherin interact withtannins, forming a signi!cant quantity of complexes (either insolubleor soluble), while bPRPs (basic PRPs) interact poorly with procyanidintrimer and gPRPs (glycosylated PRPs) only complexwith PGG. In gener-al, PGG formed a high amount of insoluble complex with salivary pro-teins while procyanidin trimer formed soluble complexes (except forstatherin). These results highlighted the in"uence and mechanisms ofdifferent tannins and salivary proteins that could present in their inter-action, and consequently in the development of the astringency sensa-tion (Soares et al., 2012).

Rawel et al. (2002) reported reductions in the amounts of lysine,cysteine and tryptophan determined in soy proteins after interactingwith different phenolic compounds. Soy glycinin and soy trypsin inhib-itor were derivatized by chlorogenic and caffeic acids (cinnamic acids,C6\C3 structure), and by gallic acid representing hydroxybenzoicacids (C6\C1 structure). Further, the "avonoids, "avone, apigenin,kaempferol, quercetin and myricetin (C6\C3\C6 structure) werealso reacted with soy proteins. They have estimated the in"uence ofthe number and the position of hydroxy substituents of these"avonoidson selected physicochemical properties of the soy proteins. The deri-vation caused a reduction of lysine, cysteine and tryptophan residuesin soy proteins. These results underlined the possible nutritional conse-quence of protein–phenolic interactions by affecting the bioavailabilityof the essential amino acids in the food systems (Rawel et al., 2002).

It has been also reported that the presence of condensed tanninsdecreased in vivo and in vitro digestibility of proteins. Their interac-tions form indigestible proteins and inhibitory digestive enzymes. Itwas observed that sorghum-condensed tannins can complex ka!rinwhich is one of the main proteins of sorghum. As a result of this com-plexation, a decrease in protein digestibility of high-tannin sorghumwas observed (Emmambux & Taylor, 2003). Duodu et al. (2003)also reviewed the factors affecting the sorghum digestibility and clas-si!ed the phenolic compounds depending on the exogenous factorsaffecting protein digestibility.

In another study, leaves harvested from Acacia drepanolobium,Acacia nilotica, Acacia seyal, Acacia tortilis, Acacia polyacantha andAcacia senegal were studied and it was observed that tannins havenegative in"uence on digestibility of proteins in vitro (Rubanza etal., 2005). Similarly, another study, on the effect of sea buckthornprocyanidins on the protein digestibility also demonstrated thatsea buckthorn procyanidins precipitated proteins and inhibited di-gestive enzymes which may have changed the digestion of proteins(Arimboor & Arumughan, 2011).

4. Effects of protein–phenolic compounds interactions onphenolic compounds

The antimicrobial activity of polyphenols has been extensively in-vestigated against a wide rande of microorganism. Among polyphe-nols, "avan-3-ols, "avonols and tannins received most attention dueto their wide spectrum and higher antimicrobial activity in compari-son with other polyphenols. They are able to suppress a numberof microbial virulence factors (such as inhibition of bio!lm formation,reduction of host ligands adhesion, and neutralization of bacterialtoxins) and show synergism with antibiotics (Daglia, 2012). Protein-polyphenol interactions may decrease the antimicrobial capacities ofpolyphenols. Von Staszewski, Pilosof, and Jagus (2011) evaluated thechanges of antimicrobial capacities of different Argentinean green teavarieties by addition of whey proteins. The results revealed some de-gree of masking in the antimicrobial activity of green tea infusionswhen whey proteins are added. The antimicrobial effects in the pres-ence of whey proteins correlated with the polyphenol content of thegreen tea infusions and increased with the reduction of whey proteinconcentration. The antimicrobial effect and potential was similarwithina pH range from 4.0 to 7.0, allowing its application to a wide group offoods (Von Staszewski et al., 2011). There are several other studies deal-ing with the changes that occur as a result of protein–phenolic interac-tions. However, in this review mainly effects on antioxidants andbioavailability 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 amatter of growing interest because it may provide a variety of infor-mation, such as resistance to oxidation, quantitative contribution ofantioxidant substances, or the antioxidant activity that they maypresent inside the organism when ingested (Huang, Ou, & Prior,2005; Serrano, Goñi, & Saura-Calixto, 2007). Numerous in vitro stud-ies have been conducted to evaluate the total antioxidant capacity(TAC) of food products. So far, however, there is no of!cial standard-ized method, and therefore it is recommended that each evaluationshould be performed under different oxidation conditions and differ-ent measurement methods. The methods for measuring antioxidantcapacity are basically classi!ed into two groups, depending on the re-action mechanism: methods based on hydrogen atom transfer andmethods based on electron transfer (Huang et al., 2005). The majorityof hydrogen atom transfer-based assays apply a competitive scheme,in which antioxidant and substrate compete for thermally generatedperoxyl radicals through the decomposition of azo-compounds. Elec-tron transfer based assays measure the capacity of an antioxidant inthe reduction of an oxidant, which results in color changes when ox-idation occurs. The degree of color change is correlated with thesample's antioxidant concentration (Zulueta, Esteve, & Frigola, 2009).

In recent years, a wide range of spectrophotometric assays hasbeen adopted to measure antioxidant capacity of foods, the most pop-ular ones are 2,2"-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid(ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, amongothers such as oxygen radical absorbance capacity (ORAC), copper re-ducing antioxidant capacity (CUPRAC) and ferric reducing antioxi-dant power (FRAP) assays. These antioxidant capacity measurementassays are used widely for all kinds of foods such as vegetables andfruits, cocoa and cocoa products, beverages including fruit juices, teaand coffee etc. There are many studies on the effect of proteins onthe antioxidant capacity of phenolic compounds in"uenced by theprotein–phenolic compound interactions. A brief outline on the effectsof protein–phenolic compound interactions on the total phenolic and"avonoid contents and total antioxidant capacities of polyphenol richfood products including the methods of analysis are given in Table 2.


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 includedboth unmodi!ed protein and the protein–EGCG adduct, increasedwith storage time at 30 °C using ABTS, FRAP and ORAC methods(Almajano et al., 2007).

Bel!"ak et al. (2009) studied the total phenolic contents, total "avo-noid contents and antioxidant capacities of various chocolate products.

They reported that the lowest total phenolic content, total "avonoidcontent and antioxidant capacities were observed in milk chocolatealthough it contains higher cocoa solids content (29%) than cocoa bars(16%). It was suggested that this decrease was as a result of strongcatechin–protein interactions. Milk based products represent a verycomplex matrix where strong catechin–protein interactions are well-known to occur and it directly in"uences catechin determination by sig-ni!cantly reducing analytical recovery from the food matrix (Bel!"aket al., 2009).

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

Product Proteins Phenolics Antioxidantcapacitymeasurementmethods

Totalphenoliccontentassays

Results Reference

Milk proteins andBSA & EGCG

Bovine serumalbumin (BSA)!-lactalbumin,"-lactoglobulin,!-casein,"-casein.

Epigallocatechin gallate (EGCG) ABTS (+) TAC Almajano et al.(2007)FRAP (+) TAC

ORAC (+) TAC

Dairy products &confectionaryproducts

Milk proteins Cocoa liquor, cocoa powder,chocolate with (88, 72, 60%) cocoasolids (cs), cooking chocolate,powdered chocolate, milk chocolate(29% cs), cocoa bar (16% cs)

TP (") TPMilk chocolate (29% cs)bcocoa bar (16%cs)

Bel!"ak, Komes,Hor#i$, Gani$, andKarlovi$ (2009)

TF (") TFMilk chocolate (29% cs)bcocoa bar (16%cs)

ABTS (") TACMilk chocolate (29% cs)bcocoa bar (16%cs)

DPPH (") TACMilk chocolate (29% cs)bcocoa bar (16%cs)

FRAP (") TACMilk chocolate (29% cs)bcocoa bar (16%cs)

Dairy products & tea Milk proteins(2% skim milk)

Tea polyphenols (Green tea,Darjeeling tea, English breakfasttea respectively)

TP (") TP Dubeau et al. (2010)ABTS (") TAC (6.0%, 8.3%, and 19.6%)Voltammetry (") TAC 4 times larger results

than ABTSLipidperoxidation

(+) TAC 19%, 10% and 12%

Dairy products & tea

(sugar+milk)

Milk proteins Tea polyphenols TP (") TP Sharma, Vijaykumar,and Jaganmohanrao(2008)

DPPH (") TACBlack tea>blacktea+sugar>blacktea+milk>blacktea+milk+sugar"-carotene-linoleic-acid assay

(+) TACDairy products & tea Milk proteins Tea polyphenols (Green and

black tea)ABTS () TAC Arts et al. (2002)

Walnut kernels Walnut proteins(mostly glutelinand globulin)

Walnut phenolics TP Whole kernelbhull Labuckas et al.(2008)DPPH Whole kernelbhull

Dairy products & tea Milk(semi-skimmedand skimmedmilk)

Tea phenolics FRAP (") TAC Ryan and Petit(2010)

Dairy products/soyamilk & tea

Semi-skimmedbovine milkSoya milk

Black tea phenolics FRAP (") TAC (semi-skimmedbovine milk+tea)0/(+) TAC (soya milk+black tea)

Ryan and Sutherland(2011)

Whey protein & tea Whey protein(8% or 16% w/v)

Argentinean black tea TP (") TP Von Staszewski et al.(2011)

Coffee & milk Milk proteins Espresso, Turkish, Instantand Filter Coffee

TP (") TP Niseteo, Komes,Bel!"ak-Cvitanovi$,Hor#i$, and Bude"(2012)

TF (") TFABTS (") TACFRAP (") TAC

Coffee & milk Milk proteins Espresso TP (") TP Sanchez-Gonzalez,Jimenez-Escrig, andSaura-Calixto (2005)

ABTS (") TACFRAP (") TAC

Coffee & tea & milk Milk proteins Tea and coffee ColoumetricFRAP method

(") TAC Ziyatdinova,Nizamova, andBudnikov (2010)

("): decrease, (+): increase, TAC: total antioxidant capacity, TP: total phenolics, TF: total "avonoids.


Studies investigating the effect of milk on antioxidant capacities oftea samples have presented contradictory results, possibly because ofthe different methods used for the measurement of antioxidant ca-pacity. Dubeau et al. (2010) measured the antioxidant capacities byusing three complementary assays; ABTS free radical scavenging,voltammetry, and lipid peroxidation inhibition. They observed thataccording to ABTS and voltammetry methods, milk decreased the anti-oxidant capacities of teas. In contrast, in the lipid peroxidation method,the results indicated thatmilk enhanced the chain-breaking antioxidantcapacity of teas. The researchers explained these !ndings as milk canhave dual effects on the antioxidant capacity of tea; an inhibitory effectfor reactions taking place in solution or at a solid–liquid interface and anenhancing effect for those in oil-in-water emulsions (Dubeau et al.,2010).

A similar interaction between the tea phenolic compounds andmilkproteins has also been reported by Sharma et al. (2008) for black tea,with sugar, milk or both added. They reported that black tea extractshad the maximum amount of polyphenol content followed by blacktea with sugar and black tea with both milk and sugar. In addition,total polyphenol contents were found to give the lowest values inblack tea with milk compared to other samples, probably due to eithercovalent or non-covalent interactions between plant phenolics and pro-teins. Arts et al. (2002) also reported that pure !, ", and #-caseinsmasked the ABTS-scavenging capacities of green and black tea extractsand of some pure "avonoids typically found in teas to different extentsprobably because of covalent or non-covalent interactions. Both inter-actions might lead to precipitation of proteins through multisite inter-actions and multidentate interactions. In multisite interactions, severalphenolics bind to oneproteinmolecule and inmultidentate interactionsone phenolic binds to several protein sites or protein molecules. Theseinteractionsmay causemasking of polyphenol contents on the proteins(Arts et al., 2002).

Labuckas et al. (2008) studied the interactions of phenolics of wal-nut kernel proteins (mostly glutelins and globulins) and their effecton total phenolic content and total antioxidant capacity using theDPPH (2,2-diphenyl-1-picrylhydrazyl) method. They reported thatthe whole kernel has a lower antioxidant capacity and total phenoliccontent compared to the hull. This is because the hull contains mostof the phenolics and, therefore, their interactions with proteins mayhave decreased the total antioxidant capacity in the whole kernel(Labuckas et al., 2008).

Ryan and Petit (2010) reported that the additions of milk to teasigni!cantly decreased the total antioxidant capacity depending onthe amount of milk compared to the additions of same volume ofwater as analyzed by the FRAP method. Besides, antioxidant activitywas decreased with increased amount of added milk. On the otherhand, Ryan and Sutherland (2011) studied the effect of adding soymilk or bovine milk on the total antioxidant capacity of !ve brands oftea. It was observed that when compared to tea with semi-skimmedbovine milk, each of the !ve brands of analyzed tea samples had signif-icantly higher total antioxidant capacities or no change after the addi-tion of soy milk. It was suggested that the addition of soy milk toblack tea might be a useful alternative to semi-skimmed bovine milkif maintaining the overall antioxidant potential of the tea infusion is de-sired (Ryan & Sutherland, 2011).

Von Staszewski et al. (2011) studied the total phenolic contents,antioxidant and antimicrobial activities of different Argentineangreen tea varieties and whey protein mixtures. Their results revealedthat when whey proteins are mixed with tea infusions, antioxidantand antimicrobial activity of green tea infusions were masked. Thedegree of inhibition of antioxidant activity in each variety did not de-pend on the total polyphenol concentration, indicating the impor-tance of the particular polyphenol composition of each variety (VonStaszewski et al., 2011).

Niseteo et al. (2012) studied the total phenolic and total "avonoidcontents and antioxidant capacity of different coffee brews and the

effects of milk addition. The results revealed that in comparison toplain brews prepared only with water, the addition of milk decreasedthe TPC and TFC of coffee and decaffeinated coffee brews, as opposedto instant cappuccino brews prepared with milk, which exhibit higherTPC when compared to plain water-made brews. Milk containing foodproducts 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 de-termination by signi!cantly reducing analytical recovery, which maybe the reason for the overestimation of milk-prepared cappuccinobrews. Contrary to the results obtained for coffee brews preparedwith milk, instant cappuccino brews made with milk exhibited higherTPC and TFC than those made with water. The higher values of TPCand TFC in cappuccino brews with milk can be explained by the pres-ence of milk derived compounds and reduced analytical accuracy, aswell as the lack of selectivity of the Folin–Ciocalteu reagent used forTPC analysis which reacts not only with phenols but also with other re-ducing compounds such as carotenoids, amino acids, sugars and vita-min C (Vinson, Su, Zubik, & Bose, 2001). Apart from instant coffee, thecomposition of instant cappuccino includes milk powder, powderedwhey proteins, lactose, sugar, vegetable fat, salt, aroma and E99 (calci-um lactobionate), ingredients that can interfere with the determinationof phenolic compounds in this assay, thus contributing to the inconsis-tency of the results. The antioxidant capacities of coffee brews obtainedby both ABTS and FRAP assays were in accordance with TPC and TFCvalues and as expected, decreased with the addition of milk. This canbe explained by the fact that up to 1/3 of the quantity of CGA, as themain 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) wasalso in agreement with the results of Niseteo et al. (2012) The re-searchers studied the effect of milk on the total phenolic content and an-tioxidant capacity of espresso samples by ABTS and DPPH assays. Theyobserved that when milk was added to the coffee, antioxidant activitydecreased depending on increasing amount of milk (Sanchez-Gonzalezet al., 2005).

Ziyatdinova et al. (2010) developed a new approach for the evalu-ation of total free polyphenols content in the presence of proteinsusing electrogenerated [Fe(CN)6]3" ions as coulometric titrant. Mix-tures of tea and coffee were prepared for further evaluating the effectof milk proteins on the antioxidant properties of beverages. Maskingeffect of milk proteins on ferric reducing power of tea and coffeere"ecting the content of free polyphenols in the systemwas 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 effectsof protein–phenolic interactions on the content of individual phenoliccompounds. These studies should be encouraged to understand theexact mechanism behind the protein–phenolic interactions.

Ferruzzi and Green (2006) studied the interactions of tea cate-chins with proteins. Their objective was to evaluate the effect of pep-sin treatment as an enzymatic step to increase catechin recovery frommilk and other formulations rich in protein. They combined brewedgreen tea with skimmed milk in ratios of 10% to 50% and analyzedtea catechins by reversed phase High Performance Liquid Chromatog-raphy (HPLC) with photodiode array detection (PDA). The results in-dicated that recovery decreased with increasing milk content. Thepresence and ratio of milk most affected gallated catechins, speci!cal-ly epigallocatechin-gallate (EGCG) and epicatechin-gallate (ECG).

Bartolome et al. (2000) prepared mixtures of BSA and several lowmolecular weight phenolics that were incubated and fractionatedusing G-50 Sephadex chromatography. Among the selected commer-cial phenolic standards tested (p-coumaric acid, p-hydroxybenzoic


acid, protocatechuic acid, caffeic acid and (+)-catechin), the strongestBSA-binding af!nity was demonstrated by 3,4-dihydroxy benzoic andcinnamic acids (protocatechuic acid and caffeic acid), whereasp-hydroxybenzoic acid did not interact with BSA. Moreover, the samemethodology was also applied to a phenolic extract obtained from len-tils containing p-coumaric acid, a p-coumaric acid derivative, (+)-cate-chin and procyanidins B3 and B1. Interactions between lentil phenolicextracts and BSA were comparable to those observed among the com-mercial standards and BSA (Bartolome et al., 2000).

According to the results of these studies, it is obvious that differentphenolic compounds might show different behaviors during their in-teractions with proteins. Taking into account that there are manytypes of phenolic compounds present in food systems, further studieson individual phenolic compounds are of critical importance to un-derstand their exact role and behavior.

4.3. Effects on in-vivo bioavailability

Proteins affect the antioxidant activity of polyphenols mostly in anegative way. They usually decrease the antioxidant activity due totheir strong binding af!nity to polyphenols. However, the effect ofprotein–polyphenolic compound interactions should also be consid-ered from the bioavailability of phenolic compounds point of view.It is important to determine whether protein interaction with poly-phenols modulate the uptake and concentration of polyphenols inplasma. A brief outline on the effects of protein–phenolic interactionson the bioavailability of phenolics is presented in Table 3.

Keogh et al. (2007) conducted experimental studies with humansubjects consisting of 24 middle-aged men and women evaluatingwhether milk proteins with cocoa polyphenols modulate the uptakeand concentration of polyphenols in plasma. It was proved that milkpowder did not in"uence the average concentration of polyphenols.Although it slightly accelerated absorption, this was of no physiolog-ical signi!cance (Keogh et al., 2007).

Duarte and Farah (2011) also studied the effect of simultaneous con-sumption of coffee and milk on the urinary excretion of CGA and metab-olites. 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 ana-lyze CGA and metabolites by HPLC/LC–MS (liquid chromatography-massspectrometry). It was observed that the amount of CGA and metabolitesrecovered after consumption of combined coffee-milk (40%±27%) wasconsistently lower in all subjects compared to that of coffee alone(68%±20%). It was suggested that the simultaneous consumption ofmilk and coffee may impair the bioavailability of coffee CGA in humans(Duarte & Farah, 2011).

In another study conducted in vivo, it was investigated whether theinteraction ofmilk reduces the bioaccessibility of tea catechins associat-ed with tea bene!cial effects. Addition ofmilk to black tea resulted withthe formation of polyphenol–protein complexes, and a decrease in totalcatechin recovery. Polyphenol–protein complexes were degraded dur-ing digestion. It was very unlikely that consumption of teawith orwith-out milk will result in a difference in catechin plasma concentration.This result is in disagreement with other studies in which a decreasein the polyphenol bioavailability was observed by the effect ofprotein–phenolic interactions (Burg-Koorevaar et al., 2011).

Kyle et al. (2007) also studied the addition of milk to tea on ab-sorption of polyphenols. It was found that consumption of black teawas associatedwith signi!cant increases in plasma antioxidant capacityand concentrations of total phenols, catechins, and the "avonols quer-cetin and kaempferol within 80 min. However, the polyphenol absorp-tion was unaffected by the addition of milk to black tea (Kyle et al.,2007).

Sera!ni et al. (2009) studied the bioavailability of phenolics and invivo antioxidant capacity of blueberries consumed with and withoutmilk by conducting the experiments with eleven healthy human

volunteers who consumed either 200 g of blueberries plus 200 mlof water or 200 g of blueberries plus 200 ml of whole milk. The re-sults showed that ingestion of blueberries increased plasma levelsof reducing and chain-breaking potential and enhanced plasma con-centrations of caffeic and ferulic acids. However, there was no in-crease in plasma antioxidant capacity when blueberries and milkwere ingested. There was a reduction in the peak plasma concentra-tions of caffeic and ferulic acid as well as the overall absorption ofcaffeic acid. As a conclusion of this study, the researchers suggestedthat milk and blueberry interactions impair the in vivo antioxidantproperties of blueberries and reduce the caffeic acid absorption(Sera!ni et al., 2009).

On the other hand, there are contradictory results about the effect ofmilk on the plasma antioxidant capacity as reviewed by Lotito and Frei(2006). It has been suggested that polypeptide chains are locatedaround the fat globule membrane, which promotes their linkage withphenolic compounds (Sera!ni et al., 2009) and probably results with adecrease in the accessibility of the colonic microbiota and thus theirdegradation (Lotito & Frei, 2006). Recently, the same behavior was ob-served in yogurt. Roowi et al. (2009) investigated the catabolism ofhesperetin-7-O-rutinoside following acute supplementation of orangejuice, with and without yogurt. The orange juice was found to containhesperetin-7-O-rutinoside and naringenin-7-O-rutinoside. GC–MS(gas chromatography–mass spectrometry) analysis of the urine sam-ples resulted with the identi!cation of nine phenolic acids, !ve ofwhich, 3-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid,dihydroferulic acid, 3-methoxy-4-hydroxyphenylhydracrylic acid and3-hydroxyhippuric acid, were relatedwith orange juice consumption sig-nifying that they were derived from colonic catabolism of hesperetin-7-O-rutinoside. The overall (0–24 h) excretion of the !ve phenolic acidsincreased signi!cantly, equivalent to 37% of the ingested "avanones, fol-lowing orange juice consumption. When the orange juice was ingestedwith yogurt, excretion fell back markedly (Roowi et al., 2009).

In another study, Green et al. (2007) characterized the effect of com-mon food additives on digestive recovery of tea catechins. Green teawater extracts were prepared containing EC, EGC, EGCG and ECG, re-spectively. Bovine, soy rice milks were added to understand the impactof creaming agents on catechin digestive recovery. Samples were ana-lyzed with in vitro digestive method which simulated gastric andsmall intestinal conditions with pre- and post- digestion catechin pro-!les measured by HPLC. Tea samples enriched with 50% bovine, soyand rice milk increased the recovery of total catechins by 52, 55 and69%, respectively. On the other hand, in vivo methods which representthe dynamic nature and heterogeneity of the gastrointestinal tract didnot give any signi!cant difference between the control sample andteas enriched with bovine, rice and soy milk. According to the results,there were no signi!cant differences found in catechin bioavailabilityand plasma antioxidant activity between tea and tea-milk beverage inhumans in in vivo observations. As a result, it was concluded that the re-sults of in vitromethods cannot be fully and directly extended to in vivoeffects (Green et al., 2007).

Arts et al. (2001) studied the interactions between human bloodplasma and quercetin, rutin, catechin and 7-monohydroxyethylrutosideusing the ABTS method. They found that the antioxidant capacity ofhuman blood plasma enriched with these phenolic compounds waslower than the sum of antioxidant capacities of both components. Thiseffect was found to be much lower in deproteinated plasma. Therefore,it was attributed to the interactions of catechols with human bloodplasma.

Leenen et al. (2000) also studied the effect of milk addition on theplasma antioxidant capacity of black and green tea samples in vivo.Blood samples were obtained at baseline and at several time pointsup to 2 h post-tea drinking. Plasma was analyzed for total catechinsand antioxidant activity, using the FRAP assay. The results indicatedthat addition of milk to black or green tea did not signi!cantly affectthe plasma antioxidant activity. In another study by Reddy et al.


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

Product Proteins Phenolics Methods Results Reference

In vivo methodsDairy products& cocoa

Milk proteins Cocoa phenolics (catechinsand epicatechins)

HPLC (Blood samples at 0, 0.5, 1, 1.5,2.3, 4, 6, 8 h, catechin (C) andepicatechin(EC) analysis)

(=) for C and EC(=) B

Keogh, McInerney,and Clifton (2007)

Dairy products& coffee

Milk proteins Coffee phenolics (CGA andmetabolites)

HPLC/LC–MS (urine samples after24 h)

(") CGA and metabolites(") B

Duarte and Farah(2011)

Dairy products& tea

Milk proteins(skimmed orfull-fat milk)

Tea phenolics (English andIndian tea catechins)

gastric, small intestinal, and brushborder digestion, total catechin(TCAT) recovery

(") TCAT(") B

Burg-Koorevaar,Miret, andDuchateau (2011)

Dairy products& tea

Milk proteins Tea phenolics (catechins,quercetin, kaempferol)

HPLC (blood samples 10 min beforethe volunteer drank one of the testbeverages, then 50, 80, and 180 minthereafter)FRAP

(=) TCAT(=) quercetin(=) kaempferol(=) B

Kyle, Morrice,McNeill, andDuthie (2007)

Fruits & tea Milk proteins Blueberry fruit phenolics Reversed phase (RP)-HPLC (") TAC(") caffeic acid(") ferulic acid(") B

Sera!ni et al. (2009)

Dairy products& fruit juices

Yogurt proteins Orange juice phenolics(hesperetin-7-O-rutinosideandnaringenin-7-O-rutinoside)

GC-MS analysis of urine (24 h) (") B of 5 phenolic acids(3-hydroxyphenylacetic acid,3-hydroxyphenylhydracrylic acid,dihydroferulic acid,3-methoxy-4-hydroxyphenylhydracrylicacid and 3-hydroxyhippuric acid)

Roowi, Mullen,Edwards, andCrozier (2009)

Dairy products& tea

Bovine, rice andsoy milkproteins (50%)

Green tea phenolicsepicatechin (EC),epigallocatechin (EGC),epigallocatechin-gallate(EGCG) andepicatechin-gallate (ECG)

HPLCpre- and post-digestion catechinpro!lesin vivo methods

(") TCAT, (") B(=) TCAT, (=) B

Green, Murphy,Schulz, Watkins,and Ferruzzi (2007)

Blood plasma& antioxidants

Human bloodplasma proteins

quercetin, rutin, (+)catechin,

7-monohydroxyethylrutoside In vivo methods, ABTS (") TAC, (") B

Arts et al. (2001)Blood plasma& antioxidants

Human bloodplasma proteins

Antioxidants (albumin,ascorbic acid, GSH, GSSG,melatonin, plasma,quercetin, uric acid)

In vivo methods, ABTS (") TAC, (") B B%auz et al. (2008)


Milk proteins Coffee phenolics (CGA) HPLC (plasma samples)In vivo methods

(=) CGA, (=) B(=) CQA, (=) B

Dupas et al. (2006)

Dairy products& tea

Milk proteins Black and green teaphenolics

In vivo methods, FRAP (=) plasma antioxidant activity Leenen, Roodenburg,Tijburg, and Wiseman(2000)

Dairy products& tea

Milk proteins Black tea phenolics In vivo methods (=) plasma antioxidant activity Reddy, Sagar,Sreeramulu, Venu,and Raghunath(2005)


Whole milkor nondairycreamer

Instant coffee phenolics In vivo methodsLiquid chromatography-Electrospray ionization-tandemMS analyses and quanti!cationof coffee phenolics in plasma

(=) caffeic acid (CA), ferulic acid (FA),and isoferulic acid (iFA) equivalents(=) CGA(=) B(=) plasma phenolics

Renouf et al. (2010)

Dairy products& tea

Milk proteins Black and green teaphenolics (quercetin andkaempferol glycosides)

HPLC (human plasma samplesevery 2 h)In vivo methods

(=) plasma phenolics(=) B

Hollman, Van HetHof, Tijburg, andKatan (2001)

Dairy products& tea

Milk proteins Black tea phenolics Flow-mediated dilation (FMD)nitro-mediated dilation (NMD)measured by high-resolutionvascular ultrasound, alsoperformed in isolatedrat aortic rings and endothelialcells (before and 2 h afterconsumption)HPLC

(") FMD(") favorable health effects of teaon vascular function(") B

Lorenz et al. (2007)

Confectionary& milk

Milk proteins Chocolate phenolics("avan-3-ols)

Human in vivo pharmacokineticsRP-HPLC

(=) B ("avan-3-ols) Neilson et al. (2009)

Dairy products& cocoa

Milk proteins Cocoa powder "avonoids LC–MS/MS (urine samples (0–6,6–12, and 12–24 h)).

Phenolic acids(") 3,4-dihydroxyphenylacetic,protocatechuic, 4-hydroxybenzoic,4-hydroxyhippuric, hippuric, caffeic,and ferulic acids(+) vanillic and phenylacetic acids

Urpi-Sarda et al.(2010)


Milk proteins Cocoa powder "avonoids LC–MS/MS (plasma samples after2 h of intake)

(=) (")-EC Roura et al. (2007)

Dairy products& chocolate

Milk proteins Chocolate "avonoids In vivo methodsFRAP

(")(")-EC Sera!ni et al. (2003)

(continued on next page)


(2005), the !ndings were in agreement with the results of Leenen etal. (2000). They investigated the effect of milk addition to black tea onthe ability to modulate oxidative stress and antioxidant status inadult male human volunteers. It did not affect the bene!cial effectsof black tea on total plasma antioxidant activity, and plasma resis-tance to oxidation induced ex vivo. It decreased plasma and urinarythiobarbituric acid reactive substance levels. The results suggestedthat the addition of milk may not obviate the ability of black tea tomodulate the antioxidant status of subjects and that consumption ofblack tea with/without milk prevents oxidative damage in vivo(Reddy et al., 2005).

In another study, eighteen healthy volunteers consumed two outof four supplements for three days: black tea, black tea with milk,green tea, and water. A cup of the supplement was consumed every2 h each day for a total of 8 cups per day. The supplements providedabout 100 $mol quercetin glycosides and about 60–70 micromolkaempferol glycosides. The addition of milk to black tea did not affectthe plasma concentration of quercetin or kaempferol. As a result, itwas concluded that "avonols were absorbed from tea but their bio-availability was not affected by the addition of milk (Hollman et al.,2001).

Renouf et al. (2010) also studied the effect of adding whole milk orsugar 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 signi!cantly alter the maximum plasmaconcentration (Cmax), or the time necessary to reach Cmax (tmax).The Cmax of caffeic acid and isoferulic acid was signi!cantly lowerand the tmax of ferulic acid and isoferulic acid was signi!cantly longerfor the sugar/nondairy creamer group than for the instant coffeegroup. In conclusion, adding whole milk did not change the overallbioavailability of coffee phenolic acids, whereas sugar and nondairycreamer affected the tmax and Cmax values but not the appearance ofcoffee phenolics in the plasma (Renouf et al., 2010).

Neilson et al. (2009) studied the effect of milk addition to choco-late on the bioavailability of EC of chocolate. EC bioavailability wasassessed from chocolate confections [reference dark chocolate(CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa bev-erages [sucrose milk protein (BSMP), non-nutritive sweetener milkprotein (BNMP)], in humans. These results suggested that the pres-ence of milk solids did not signi!cantly change the bioavailability ofEC among chocolate confection products (Neilson et al., 2009).

Urpi-Sarda et al. (2010) studied the effect ofmilk on the bioavailabilityof cocoa "avonoids considering phase II metabolites of epicatechin. Theystudied the effect of milk at the colonic microbial metabolism level ofthe non-absorbed "avanol fraction that reaches the colon and is metabo-lized by the colonic microbiota into various phenolic acids. Phenolic acids

were analyzed by LC–MS/MS after solid-phase extraction. Of the 15 me-tabolites assessed, the excretion of 9 phenolic acidswas affected by the in-take 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, whereasurinary concentrations of vanillic and phenylacetic acids increased. Inconclusion, milk moderately affected the formation of microbial phenolicacids derived from the colonic degradation of procyanidins and othercompounds 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.Quanti!cation of (")-EC in plasma was determined by LC–MS/MSanalysis after a solid-phase extraction procedure. Milk solution, asone of the most common ways of cocoa powder consumption,seems to have a negative effect on the absorption of polyphenolsbut it was not statistically signi!cant (Roura et al., 2007).

There are many other studies on the bioavailability of polyphenolsfrom cocoa that have yielded contradictory results. Sera!ni et al.(2003) suggested that interaction between milk proteins and choco-late "avonoids inhibits the absorption of (")-epicatechin ((")-EC)into the bloodstream. On the other hand, Schroeter et al. (2003)showed that there was no signi!cant difference in the (")-EC con-centration in the plasma after the consumption of a milk-containingor water-based cocoa beverage under isocaloric and isolipidemicconditions. Schramm et al. (2003), after evaluating the effect of sever-al foods including sugar, milk, bread, steak, and grapefruit on the ab-sorption and pharmacokinetics of cocoa "avanols, also concluded thatmilk 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 etal. (2006) studied the effect of milk addition to coffee on the bioavail-ability of coffee phenolics, mainly CGA. Interactions between CGA andmilk proteins were investigated using an ultra!ltration technique.These interactions proved to be slightly disrupted during an in vitrodigestion process. CGA absorption and bioavailability were then stud-ied in vitro using a Caco-2 cell model coupled with an in vitro diges-tion process. The results revealed that CGA absorption under itsnative form is weak, and unmodi!ed 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 phe-nolics and milk proteins, but there was no signi!cant effect observedon the bioavailability of CGA (Dupas et al., 2006). On the other hand,Neilson et al. (2009) studied the effect of milk addition to chocolate

Table 3 (continued)

Product Proteins Phenolics Methods Results Reference

In vivo methodsDairy products& cocoa

Milk proteins Cocoa beverage "avonoids In vivo methodsHPLC

(=) (")-EC Schroeter, Holt,Orozco, Schmitz,and Keen (2003)


Milk proteins Cocoa "avonols In vivo methodsHPLC

(=) "avonols Schramm et al.(2003)

In vitro methodsDairy products& coffee

Milk proteins Coffee phenolics (CGA) Caco-2 cell model coupled with an invitro digestion process

(=) CGA, (=) B(=) CQA, (=) B

Dupas et al. (2006)

Fruits & bovineserumalbumin

Bovine serumalbumin

Sea buckthornproanthocyanidins

In vitro digestion (") B Arimboor andArumughan (2011)

Confectionary& milk

Milk proteins Chocolate phenolics("avan-3-ols)

In vitro DigestionCaco-2-cell culture experiments

(=) B ("avan-3-ols) Neilson et al. (2009)

Dairy products& tea

Milk proteins(casein and BSA)

Black and green teaphenolics (quercetin,rutin)

Colourimetric method by usingelectrogenated bromine

(") B Nizamova, Ziyatdinova,and Budnikov (2011)

(=): no signi!cant difference, (+): increase, ("): decrease, B: bioavailability of phenolics, TCAT: total catechins, TAC: total antioxidant capacity.


on the bioavailability of EC of chocolate. According to the results, invitro bioaccessibility and Caco-2 accumulation did not differ betweentreatments. Bioavailability as measured by the areas under the serumconcentration–time curve (AUC) was not signi!cantly differentbetween confections which is in agreement with the studiessuggesting that the presence of milk does not negatively affect thebioavailability of cocoa "avan-3-ols (Neilson et al., 2009).

Arimboor and Arumughan (2011) studied the effect of sea buck-thorn proanthocyanidins on in vitro digestion of proteins. It wasfound that interactions of sea buckthorn proanthocyanidins withfood proteins and digestive enzymes might alter the protein digest-ibility and phenolic bioavailability, negatively. In another study,Nizamova et al. (2011) proposed a new method for the estimationof the bioavailability of polyphenols using electrogenerated bromineas a coulometric titrant. The titration of model solutions of caseinand BSA showed that casein did not interact with electrogeneratedbromine, while BSA reacted with the titrant in the ratio of 1:63. Theproteins bound rutin and quercetin at a high rate and thus reducedthe bioavailability of polyphenols. The concentration of free polyphe-nol was reduced with an increase in the concentration of protein inthe mixture. The total antioxidant capacity of tea samples was alsodetermined, and it was reported that green tea possess higher totalantioxidant capacity than the black one because of the partial oxida-tion of polyphenols to respective thearubigins and thea"avins at thefermentation step in the production of green tea. According to the re-sults, the total antioxidant capacity of tea decreased with the amountof milk. Milk proteins bound tea polyphenols into complexes as a re-sult of intermolecular interactions and reduced their bioavailability,accordingly (Nizamova et al., 2011).

5. Discussion

As a result of protein–phenolic interactions changes occur both onthe proteins and phenolic compounds. The main changes observed inproteins are basically related with the structure, functional and nutri-tional properties, and digestibility of proteins. On the other hand,these interactions result with changes in total phenolic or "avonoidcontents, antioxidant activities, contents of individual phenolic com-pounds, as well as in-vitro and in-vivo bioavailability of phenoliccompounds. Conditions such as temperature, pH, types of proteins,protein concentration, types and structures of phenolic compoundsand several other factors affect protein–phenolic interactions. Thereare some contradictory results about the effect of temperature onthe binding af!nity of polyphenols to proteins. Some studies indicat-ed that binding af!nity decreases by increasing temperature whereassome others reported vice versa. Unlike the temperature, thereare more comparable results regarding the effects of pH on protein–phenolic interactions. Lower pH results with a stronger bindingsince the dissociation of protein leads to more binding sites at lowerpH values. On the other hand, the different hydrophobicity and iso-electric points of proteins, and the difference in the amino acid com-position lead to differences in the binding af!nity of phenoliccompounds to some proteins. The protein concentration in the solutionaffects the protein–phenolic interactions, thus the precipitation is de-creased after high concentrations reached in the solution. Differenttypes and structures of phenolic compounds also affect protein–pheno-lic interactions. Some of the structural elements that in"uence the af!n-ities of polyphenols for proteins are the following (Xiao et al., 2011): (i)Methylation andmethoxylation of "avonoids decreased or only slightlyaffected the af!nities; (ii) hydroxylation on rings A and B of "avonesand "avonols slightly enhanced the interaction and hydroxylation onthe ring A of "avanones and signi!cantly improved the af!nities; (iii)glycosylation of polyphenols weakened the af!nities; (iv) hydrogena-tion of the C2_C3 double bond of "avonoids decreased the bindingaf!nities (v) galloylation of catechins and esteri!cation of gallic acidsigni!cantly improved the binding af!nities. There are also other factors

that in"uence the protein–phenolic interactions such as salt concentra-tion and addition of certain reagents. However, the number of studiesinvestigating the effect of all these parameters is very limited andshould be further studied in more detail to optimize the process condi-tions and to improve the bene!cial health effects of phenolics. Processconditions and products can then be designed in away that will providethe maximum bene!cial health effects to the consumers, and also opti-mum nutritional and functional properties can be supplied to theproducts.

Protein–phenolic interactions in"uence the structure, functionaland nutritional properties, and digestibility of proteins. It was ob-served that the protein–phenolic interactions increase the molecularweight of proteins. The presence of phenolic compounds also affectsprotein solubility which is an important factor in protein functionali-ty, because protein insolubility also hinders other protein functionalproperties. The increase in the content of phenolic compounds de-creases the solubility of proteins. In addition, interaction of proteinswith phenolic compounds may improve the thermal stability of pro-teins. Moreover, proteins may also lead to some undesirable changesin color and taste when they interact with phenolic compounds. Fur-thermore, looking from the nutritional value and digestibility point ofview, it was observed that protein–phenolic interactions decrease thenutritional value of some essential food components and also in vivoand in vitro digestibility of proteins.

On the other hand, some other studies focused on the changes inphenolic compounds as a result of protein–phenolic compound interac-tions and investigations on the changes in antioxidant capacities andbioavailability of phenolic compounds were performed. However, re-search on individual phenolic compounds is lacking. On the otherhand, although the bioactivity of a molecule mainly depends on thefunctional group/moiety in themolecule, the differences in themolecu-lar structure were not considered in the studies performed so far. It isimportant to understand the involvement of the functional groups orthe position of these functional groups in protein–phenolic complex,and it should be investigated in detail to better understand the mecha-nisms taking place.

Another important drawback of the protein–phenolic interactionstudies is that they are generally performed using only tea, coffee,and chocolate as phenolic compound sources and milk as the proteinsource. Tea and coffee are the most widely consumed beverages inthe world with a high antioxidant capacity and they are often con-sumed together with milk. Therefore, it is important to understandthe effect of milk proteins on the antioxidant capacity and bioavail-ability of coffee and tea phenolics, as they also account for most ofthe polyphenol daily intake in the countries where the fruits and veg-etables are not preferred to be eaten very often. However, it is still ofcritical importance to work on other food proteins such as plant-originated proteins, to understand the effects more clearly and to beable to compare the effect of different protein sources on these inter-actions. On the other hand, a better understanding of the protein–phenolic interactions for a large variety of food products may providebene!t for improving the process conditions and parameters for thefood products that contain both proteins and phenolic compounds.This information can also help developing new food products withbetter nutritional quality and improved health aspects.

According to many of the studies reviewed in this paper, it can beconcluded that proteins signi!cantly decrease the antioxidant capac-ity in general. There are some contradictory results between thesestudies which may arise from different methodologies used to mea-sure the antioxidant capacity or total phenolic/"avonoid contents. Itis expected that a single method cannot determine all the antioxidantcompounds available in the food matrix correctly. All the methodshave their own advantages and disadvantages. Even the results ofthe methods sharing the same principle like ABTS and DPPH canshow important differences in their response to antioxidants. Similar-ly, the extraction procedure used during analysis needs special


attention as the stability of protein–phenolic complex might be dif-ferent in different solvent systems. As mentioned above, there arefew studies about the effect of phenolic-protein interactions on indi-vidual phenolics. Most of the studies measured the total content ofphenolics, and "avonoids or the total antioxidant capacity, but thereis still a need for more studies that investigate individual phenolicsto understand the mechanism better. Especially, use of more compre-hensive methods such as LC–MS will provide more detailed informa-tion on the interactions of proteins and phenolic compounds.

Contradictory results are observed also in the bioavailability stud-ies. Some studies indicated that protein–phenolic compound interac-tion decreases the bioavailability, others reported no signi!cantchanges and a very few revealed that it increases the bioavailabilityof some phenolic compounds. This fact might be due to a high vari-ability in the absorption of "avanols in humans, as well as to thesmall number of subjects selected in the studies. Thus, bioavailabilityof phenolics should be studied in a homogenous population includinga greater number of subjects.

It is also of critical importance to evaluate the bioavailability ofhealth associated compounds present in these food materials, whichwill provide valuable data for elucidating the true biological relevanceof these compounds in the context of nutrition and human health.Bioavailability differs greatly from one polyphenol to another, andfor some compounds it depends on the dietary source. So, it is impor-tant to work with different food sources of proteins and phenoliccompounds to better understand the exact effect of interactions ofthese two compounds. According to the results of some bioavailabili-ty studies, in vitro methods can be well correlated with the results ofin vivo experiments (Bouayed, Hoffman, & Bohn, 2011). In vitro di-gestion and dialysis methods for simulating the gastrointestinal (GI)digestion are being extensively used since they are rapid, safe, anddo not have the same ethical restrictions as in vivo methods (Lianget al., 2012). On the other hand, some researchers reported that theresults of in vitro methods cannot be fully and directly extended tothe results of in vivo methods (Green et al., 2007). So, the interactionof proteins and phenolic compounds would be better evaluated byboth in vitro and in vivo models to provide comparable and more ac-curate results.

6. Conclusions

In summary, polyphenols which have been widely studied fortheir health promoting and disease preventive activities in humansare well-known to have high af!nity to bind proteins. Therefore, inter-action of phenolics and proteins affect both of these food constituents infood systems. However, the mechanisms and the consequences of theirinteractions should be studied extensively due to the fact that contra-dictory results were obtained so far. Although, there are several studiesinvestigating the effect of protein–phenolic interactions on the antioxi-dant capacities and bioavailability of phenolic compounds, research onindividual phenolic compounds is lacking. On the other hand, under-standing the exact mechanism for a large variety of food productsmay provide bene!ts for improving the process conditions and pa-rameters for the food products that contain both proteins and phe-nolic compounds. Outcomes of these studies can help devolop newfood products with better nutritional quality and bene!cial healthaspects.

It can be concluded that proteins signi!cantly decrease the antiox-idant capacity in general, but there are some controversies about theresults observed by different antioxidant capacity methods which isprobably due to the differences in the principles or fundamentals ofthese methods. That's why it is recommended to use several testmethods and compare the results obtained. In addition, more com-prehensive methods such as LC–MS would better be used to obtainmore detailed information on the interactions of proteins and pheno-lic compounds. Results of bioavailability studies can be strengthened

by using a homogenous population including a greater number ofsubjects. Further work on this topic is still necessary to clarify thecontroversial results obtained so far and to better understand themechanisms underlying protein–phenolic interactions as well as fac-tors affecting the degree of this interaction.

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