The interaction of cocoa polyphenols with milk proteins studied by

Food Research International 54 (2013) 406415

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The interaction of cocoa polyphenols with milk proteins studied byproteomic techniques

Monica Gallo a, Giovanni Vinci b, Giulia Graziani b, Carmela De Simone b, Pasquale Ferranti b,c,a Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via Pansini 5, 80131 Naples, Italyb Department of Agriculture, University of Naples Federico II, via Universit 100, 80055 Portici, Naples, Italyc Institute of Food Science, National Research Council, via Roma 52, 83100 Avellino, Italy

Corresponding author at: Department of AgricultuTel.: +39 081 2539359; fax: +39 081 7762580.

E-mail address: [email protected] (P. Ferranti).

0963-9969/$ see front matter 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.foodres.2013.07.011

a b s t r a c t
a r t i c l e i n f o
Article history:Received 30 March 2013Accepted 3 July 2013

Keywords:CocoaPolyphenolsAntioxidant activityMass spectrometryMilk proteins

The molecular interaction of cocoa polyphenols with milk proteins were investigated in vitro by combinedproteomic and biochemical strategies. Mass spectrometry and antioxidant activity assays allowed monitoringthe binding of casein and whey protein fractions to cocoa polyphenols. In particular, the nature of interactionof -lactoglobulin (-Lg) with catechin and epicatechin was characterized and the amino acid residue at thebinding site was identified. On the other side, antioxidant activity assays also showed a significant effect of thevarious milk protein fractions in decreasing the in vitro antioxidant activity of polyphenols, suggesting theexistence of other types of proteinpolyphenol interactions, probably weaker non-covalent bonds. From a nutri-tional point of view, these data indicate that the -Lg covalent modification by polyphenol alone do not supportthe hypothesis of a decrease in the bioavailability of polyphenols themselves (Scalbert &Williamson, 2000). Thismight also explain themaintenance of the antioxidant properties of cocoa polyphenols in cocoa-basedbeverages.These results suggest the perspective use of the model system developed to study other complex food matrices.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Phenolic compounds are one of the most represented groups ofsubstances in the plant kingdom: there are currently more than8000 known phenolic structures produced by secondary metabolismof plants (Bravo, 1998). Among these compounds, phenolic acids andflavonoids account for 30% and 60% of total polyphenols ingestedwith diet, respectively (Manach, Scalbert, Morand, Rmsy, & Jimnez,2004; Ramos, 2007; Xiao et al., 2011). Phenolic compounds are raisinggreat interest inmedical and scientific research for their health benefits,which include anti-carcinogenic, anti-atherogenic, anti-inflammatory,anti-microbial, anti-hypertensive activities (Gryglewski, Korbut, Robak,& Swies, 1987; Kaul, Middleton, & Ogra, 1985; Mascolo, Pinto, &Capasso, 1988).

Until now, the polyphenols in green tea, black tea, grape and wine(especially red) have been extensively studied and characterized. Onlyrecently attention has also focused on the characterization of the phenolcomponents of cocoa (Snchez-Rabaneda et al., 2003; Zumbea, 1998).Cocoa beans are a rich source of polyphenols contributing to about10% of the dry weight of the whole seed. Cocoa polyphenols have ashort half-life in plasma and a rapid excretion rate and, therefore,have a relatively low bioavailability (Manach, Williamson, Morand,Scalbert, & Rmsy, 2005). Among the different classes of flavonoids,

re, University of Naples, Italy.

ghts reserved.

procyanidins appear to be from 10 to 100 times less absorbed of theirmonomeric constituents (Tsang et al., 2005).

If assumed daily in the diet, beneficial effects of phenolic compoundscanbe cumulative (Cooper, Donovan,Waterhouse, &Williamson, 2008),especially at high doses (Heiss et al., 2007). However, Manach et al.(2005) suggested differences in individual adsorption of polyphenolsprobably linked to specific polymorphisms. This could explain thehigh variability in reported flavonoid absorption rates in vivo(Lamuela-Ravents, Romero-Prez, Andrs-Lacueva, & Tornero,2005). Absorption and metabolism of polyphenols are determinedby their chemical structure, which is correlated with the degree ofglycosylation, acylation, polymerization, conjugation with otherphenolic compounds, molecular size and solubility (Bravo, 1998).

When ingested, the first stage in the interaction between foodpolyphenols and proteins occurs in the mouth, where flavanols reactfirst with proline-rich salivary proteins forming insoluble complexesresponsible for the perception of astringency and for the characteristictaste of various food products (e.g. fruit, cocoa, coffee, tea, beer andwine) (Baxter, Williamson, Lilley, & Haslam, 1996; Jobstl, O'Connell,Fairclough, Mike, & Williamson, 2004).

In some foods, proteins and polyphenols combine to form solublecomplexes, which can reach colloidal size, causing turbidity of bever-ages and limiting the shelf life of these products. In the beer, wine andjuice production, for example, polyphenols may induce formation ofprotein precipitates with the appearance of negative sensory character-istics for the consumer (Baxter, Lilley, Haslam, & Williamson, 1997;Siebert, 1999; Siebert, Carrasco, & Lynn, 1996a; Siebert, Troukhanova,

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407M. Gallo et al. / Food Research International 54 (2013) 406415

& Lynn, 1996b). Polyphenols, in general, interact with globular proteinsand can cause structural and conformational changes of the protein. It hasbeen shown that binding affinity depends on the molecular size of thepolyphenol molecule: the higher the molecular size of the polyphenol,the greater the tendency to form complexes with proteins (De Freitas &Mateus, 2001).

Structural changes in globular proteins have already been investi-gated in milk/tea mixtures. Interaction of milk proteins with tea poly-phenols induces structural changes in both whey proteins and caseins(Hasni et al., 2011; Hudson, Ecroyd, Dehle, Musgrave, & Carver, 2009;Jobstl, Howse, Fairclough, Mike, & Williamson, 2006; Kanakis, Hasni,Bourassa, Polissiou, & Tajmir-Riahi, 2011). These changes may explainthe effect of milk addition on the antioxidant activity of tea as well asother food polyphenols (Stojadinovic et al., 2013). However, despitethe numerous studies, the binding mechanism of tea polyphenols withmilk proteins has neither been clarified yet, nor is it known whetherthis interaction is preferential to certain amino acids (Stanner, Hughes,Kelly, & Buttriss, 2004). With regard to the interactions between milkproteins and polyphenols of cocoa and chocolate, it has been shownthat, following intake of chocolate, the absorption of epicatechin(one of the major cocoa flavanols) is very low, and is still lower ifcocoa is assumed together with milk. This suggested that milkproteins sequester cocoa polyphenols limiting their adsorption inthe gastrointestinal tract (Serafini et al., 2003). However, data inthe literature are still conflicting, as later studies have found noreduction in the bioavailability of epicatechin when cocoa wasconsumed with milk (Schroeter, Holt, Orozco, Schmitz, & Keen,2003). In the light of these contrasting reports on the bioavailabilityof cocoa polyphenols in the presence of milk, the purpose of this studywas to investigate the interaction between polyphenols and proteins incocoa/milk systems, by means of complementary mass spectrometrytechniques. A simplified model system was then developed, in whichthe polyphenols were incubated with isolated milk protein fractions.The time-dependent proteinpolyphenol in vitro interactions werestudied by either MALDI-TOF or ESI mass spectrometry, showing amajor reactivity of -lactoglobulin (-Lg) towards cocoa polyphenols.The data acquired allowed us to characterize the molecular basis of theinteraction between themain cocoa flavanols (catechin and epicatechin)with -Lg by MS/MS structural analysis. The effect of non-enzymaticglycosylation, always occurring in commercial milkcocoa products dueto thermal treatments, on -Lg interaction with polyphenols wasstudied. These data were complemented by total antioxidant capacityassays on the proteinpolyphenol mixtures in order to correlatepolyphenol activity changes to structural interactions with proteins.Finally, the model system in vitro results were verified on a commercialchocolate/milk beverage.

2. Materials and methods

2.1. Reagents and standards

The phenol standards catechin (CAT), epicatechin (EPI), procyanidins,-Lg, ferulic acid, caffeic acid, coumaric acid, protocatechuic acid,chlorogenic acid, gallic acid as well as trypsin (proteomic grade), 6-hydroxy-2,5,7,8-tetramethylchromano-2-carboxylic acid (Trolox), theammonium salt of 2,2-azinobis-(3 ethylbenzothiazoline-6-sulfonicacid) (ABTS), potassium persulfate (K2O8S2) and formic acid weresupplied from Sigma-Aldrich (Italy). Oligomeric proanthocyanidins(OPCs) were purified as previously reported (Rigaud, Escribano-Bailon,Prieur, Souquet, & Cheynier, 1993). -Cyano-4-hydroxycinnamic acid(-cyano), 3.5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) and2.3 dihydroxybenzoic acid (DHB) were supplied by Fluka (Italy).Ultrapure water and all solvents of chromatographic purity werepurchased from Romil (Italy). Hydrochloric acid and Folin reagentwere supplied by Riedel de-Han (Seelze, Germany), ethanol andtrifluoroacetic acid were supplied from Carlo Erba (Italy).

2.2. Phenol extraction, quantitation and characterization by LC/MS/MS

A commercial sample (1 g) of defatted cocoa powder (Perugina, Italy)was extracted with 10 mL of methanol/water (70/30 v/v) in ultrasonicbath for 1 min. The sample was centrifuged at 4000 rpm for 10 minand the supernatant defattedwith 10 mLhexane. The polyphenol extractwas centrifuged at 13,000 rpm for 3 min and the supernatant filteredwith Phenex filters RC 0.22 m.

Total phenol concentration in the different extracts were determinedspectrophotometrically by the FolinCiocalteu assay (Box, 1983) usinggallic acid as a standard. An aliquot of 125 L of each extract was mixedwith 125 L of FolinCiocalteu phenol reagent and reaction carried outfor 6 min. Then, 1.25 mL of saturated Na2CO3 solution (7.5%) wasadded and allowed to stand for 90 min before the absorbance of thereaction mixture was measured in triplicate at 760 nm. The total phenolcontent was expressed as mg gallic acid equivalents/mL of sample.

The identification of polyphenol compoundswas performed by liquidchromatography coupled with tandem mass spectrometry (LC/MS/MS).Chromatographic separations were obtained using a HPLC equippedwith two micropumps series 200 (Perkin Elmer, Canada) and a ProdigyODS3 100 column (250 4.6 mm, particle size 5 M) (Phenomenex,CA, USA). The eluents were: A, water 0.2% formic acid; B, CH3CN/MeOH(60:40 v/v). The gradient used was as follows: 2030% B (6 min), 3040% B (10 min), 4050% B (8 min), 5090% B (8 min), 9090% B(3 min), 9020% B (3 min) at a constant flow of 0.8 mL/min. A flow of0.2 mL/min was sent into the mass spectrometer. The injection volumewas 20 L. MS and MS/MS analyses of cocoa extracts were performedwith an API 3000 triple quadrupole (Applied Biosystems, Canada) instru-ment equipped with a TurboIonspray source. Acquisition was performedin negative ion mode by multiple reaction monitoring (MRM).

2.3. Extraction of milk proteins

Raw cowmilkwas defatted by centrifugation at 5000 rpm for 30 minat 4 C. Separation of the casein fractions was carried out by isoelectricprecipitation according to Aschaffenburg & Drewry (1957). Casein waslyophilized and stored for subsequent experiments. Whey proteins(WP)were precipitated from themilkwheywith 12% (v/v) trichloroace-tic acid (TCA). The pellet was recovered after centrifugation at 4500 rpmfor 30 min and washed three times with acetone at20 C for 30 min,and finally centrifuging at 4500 rpm for 30 min. The dry residue wasused for analysis.

2.4. RP-HPLC analysis of whey proteins

HPLC analysis of WP was performed using a modular HP 1100(Agilent) instrument equipped with a C4 Reverse Phase 5 m;250 mm 2.1 mm (Vydac, 218TP54) column. Solvent A was 0.1%trifluoroacetic acid (TFA) in H2O and solvent B 0.1% TFA in acetonitrile(ACN). The chromatographic separationwas carried outwith a B gradientfrom 35 to 55% in 65 min, at the constant flow of 1 mL/min. The eluatefrom the column was monitored 220 and 280 nm. For analysis of WP,150 L sample dissolved at a concentration of 2 mg/mL in H2O/TFA 0.1%were injected. The chromatographic peaks containing -lactalbumin(-La) and -Lg were dried, freeze-dried and finally stored in a freezerat20 C.

Lactosylated -Lg (Lac--Lg) was isolated by RP-HPLC separation inthe same conditions as a commercial sample of pasteurized whole milk.

2.5. Study of in vitro interaction of polyphenols with caseins and WP

Interactions between polyphenol components of cocoa and milkproteins were studied using a simplified model obtained by incubatingmilk individual caseins and WP with cocoa polyphenols extracts andwith polyphenol standards. Proteins were solubilized in 5 mM ammo-nium bicarbonate (AMBIC) at pH 6.8 (close to milk pH of the milk), at

408 M. Gallo et al. / Food Research International 54 (2013) 406415

concentrations of 5.4 mg/mL, 5.7 mg/mL, and 2.9 mg/mL for caseins,whey proteins and -Lg, respectively. The cocoa polyphenol extractand individual polyphenols were dissolved in methanol and addedat a 10-fold molar excess compared to that of protein. Proteinpolyphenol mixtures were incubated at 37 C and the interactionmonitored for 048 h by antioxidant activity assays and massspectrometry analysis.

2.6. Alkylation and tryptic hydrolysis of -Lg

Alkylation of -Lg (IAA--Lg). -Lg (3 mg/mL) was dissolved in5 mM ammonium bicarbonate, pH 8.4, containing iodoacetamide (IAA)in molar ratio 10:1, to obtain alkylation of free cysteine residues. Thereactionwas carried out for 45 min in the dark. IAA excess was eliminat-ed by diafiltration with 3 kDa cut-off filters Centricon (Amicon, Italy).

Trypsin hydrolysiswas carried out by adding proteomic-grade trypsinand incubating at 37 C for 4 h.

2.7. MALDI-TOF-MS analysis

MALDI-TOF-MS experiments were carried out on a Voyager DE-Pro(Applied Biosystems, Framingham, USA) instrument. Spectra wereacquired either in linear or reflector mode using the delayed extractiontechnology; external calibration was performed with a mixture ofpeptides standard low molecular masses. Spectra of proteins incubatedthe polyphenols were acquired by loading 1 L of the protein sampleand using sinapinic acid (10 mg/mL in 50% ACN/TFA0.1%) as matrix. Forpeptide analysis, 1 L of peptide mixture was loaded on the appropriatetarget steel using -cyano acid as matrix (10 mg/mL in 50% ACN/TFA0.1%). MS/MS analysis of selected ions was made with Post-SourceDecay (PSD). The identification of the peptides was performed bycomparing the measured m/z values with those obtained by simulatingthe theoretical tryptic digestion of -Lg using General Protein/MassAnalysis for Windows software (GPMAW).

2.8. ESIMS/MS analysis

ESIMS/MS data were obtained using a Q-STAR MS (AppliedBiosystems, Foster City, CA) equipped with a nanospray interface(Protana, Odense, Denmark). The dried samples were resuspended inH2O and TFA 0.1%, were purified by the use of micro ZipTip C18 columns(Millipore, Milan, Italy), and sprayed through gold-coated borosilicatecapillaries (Protana). The capillary voltage used was 800 V. The collisionenergy ranged from 20 to 40 eV, depending on the size of the peptide.The collision induced dissociation spectra were processed using theAnalyst 5 software (Applied Biosystems). LC/ESI-MS analysis of -Lgtryptic peptides was carried out using a C18 reversed-phase column(218TP52, 5 m, 250 2.1 mm, Vydac, Hesperia, CA, USA) with aflow rate of 0.2 mL/min on an Agilent MSD 1100 system. Solvent Awas 0.03% TFA inwater, and solvent Bwas 0.02% TFA in ACN. Separationwas carried out in a linear gradient of 570% solvent B for 60 min. MSacquisition was in positive selected ion monitoring mode.

2.9. Antioxidant activity assays

Antioxidant activity was determined on samples prepared as de-scribed in paragraph 2.5 using the ABTS method (Pellegrini et al.,2003). In particular, different samples were opportunely dilutedwith methanol, centrifuged for 5 min at 4000 rpm and used for thedetermination of antioxidant activity. The absorbance at 734 nm ofthe ABTS discolored solution was recorded on a SpectrophotometerUV/VIS UV2100 (Shimadzu, Japan). The values were expressed inmmol equivalent of Trolox/L.

2.10. Statistical analysis

Data are expressed as mean (SD). Significance of differences wasassessed by one way analysis of variance (ANOVA) and (when theF value was significant) by the TukeyKramer test for multiple com-parisons or by Student's t test for comparison between two means.Differences were considered to be significantly different if p b 0.05.

3. Results

3.1. Analysis of phenolic components in cocoa by LC/ESIMS/MS

As a preliminary step to study of interaction of polyphenols withmilk proteins, the polyphenol composition of the commercial cocoasamples uses in this study was determined by LCMS/MS (notshown). The TIC chromatogram of cocoa polyphenol extract showedall the expected compounds previously described in cocoa (Cooperet al., 2007; Hammerstone, Lazarus, Mitchell, Rucker, & Schmitz,1999), including catechin and epicatechin and oligomeric procyanidins.Minor amounts of protocatechuic acid, chlorogenic acid and glucosideesters of apigenin, naringenin, kampferol, luteolin and quercetin werealso detected. Because of the complexity of milk/cocoa mixtures, a sim-plified model study was preliminary set up mixing single polyphenolcompounds (catechin, epicatechin, and oligomeric proanthocyanidins)as well as with those extracted from the cocoa powder with isolatedcow milk protein fractions (casein, whey protein and -Lg). Themixtures were incubated at 37 C for times ranging from 0 to 24 hand aliquots were taken at time intervals for MS analysis and antioxi-dant assays.

3.2. Antioxidant activity of polyphenol compounds

To set up the optimal conditions to measure antioxidant activity,ABTS assays (Huang, Ou, & Prior, 2005) were preliminarily carriedout testing solutions of pure catechin, epicatechin, OPCs (oligomericproanthocyanidins) and polyphenol extracts from cocoa.

The ABTS assay results (Fig. 1a) showed that after 4 h incubation at37 C, pH 7.4, polyphenol samples showed no significant changes oftotal antioxidant capacity. This indicated that the incubation conditionsdid not introduceundesiredpolyphenol oxidation.OPCs showed a slightbut significant (p b 0,05) increase of antioxidant activity, possibly amere effect of their slow solubilization process, or a consequence ofthe time/temperature conditions for incubation which induced slowconversion to large-sized polymers with consequent increase of antiox-idant capacity. Actually, it has been shown that aged wine, in which thepolymerization of polyphenols to tannins increased antioxidant capaci-ty (for a review see Cheynier, 2005). Total polyphenol measurement(Fig. 1b) confirmed the results of ABTS assay. No quantitative variationof total polyphenol content was observed after 24 h. Therefore, basedon both ABTS and Folin data, it was possible to conclude that the assayconditions did not affect the polyphenol antioxidant activity.

3.3. Total antioxidant activity of polyphenol/protein mixtures

To measure changes of total antioxidant capacity determined bypolyphenol/protein interaction, ABTS assays were performed afterincubation of casein, whey proteins or-Lgwith individual polyphenolsand total polyphenols extracted from cocoa.

Fig. 2 shows the antioxidant activity by ABTS assay as for polyphenolsincubated with the purifiedmilk protein fractions. In the mixture casein/polyphenol (Fig. 2a), the antioxidant activity decreased significantly(p b 0,05) after 24 h of incubation, thus showing a significant effect ofcasein on polyphenol activity. This decrease was more pronounced inthe case of casein incubated with either catechin or epicatechin.

These resultswere in agreementwith previous studieswhich showedthat incubation of polyphenols affected casein structure, causing a

Fig. 1. (a) ABTS assay for standards and polyphenol extracts from cocoa powder (values are expressed asmillimoles of Trolox/L). (b) FolinCiocalteu assay of polyphenol standards and ofthe extracts from cocoa powder (values are expressed as mg gallic acid/mL). Mean (SD) from three separate experiments run in duplicate. *p b 0.05 versus t = 0.


reduction of-helix and -sheet structures and an increase of unfolding(Hasni et al., 2011; Hudson et al., 2009; Jobstl et al., 2006), thussuggesting the existence of interaction with phenols.

Fig. 2bc shows the results of ABTS assay after incubation of individualphenols with whey proteins and with purified -Lg, respectively. In bothcases, the trend of the antioxidant activity after 24 h of incubation wassimilar to that observed for caseins, suggesting also in this case thatprotein/polyphenol interaction reduced antioxidant activity of thepolyphenol system by subtracting free polyphenols. Interestingly, thisdecrease was more pronounced in the case of -Lg alone. These datawere in good agreement with previous reports (Stojadinovic et al.,2013).

3.4. Characterization of protein/polyphenols interactions by MALDI-TOF-MS

The mixtures previously analyzed for antioxidant activity werecharacterized by mass spectrometry. The time-dependent formationof covalent proteinpolyphenol complexes was first investigated byMALDI-TOF-MS on the basis of the protein mass increase.

MALDI-TOFmass spectra of incubated caseins did showany stable ad-duct between the individual caseins neither with catechin/epicatechinnor with extracted cocoa polyphenols.

In the system whey protein/polyphenol, mass spectra showed noreactivity of -La with extracts of cocoa polyphenols, catechin orepicatechin over 2448 h incubation time (Fig. 3). The same spectra,instead, showed a significant reactivity for -Lg (both variants A andB, molecular mass 18,367 Da and 18,281 Da respectively) towardscatechin or epicatechin, either pure or present in the cocoa extract.In fact, after incubation with the cocoa polyphenols (Fig. 4), a288 Da mass increase (18,655 Da for variant A and 18,569 Da forvariant B) was observed in the protein molecular mass, correspond-ing to addition of a single epicatechin or catechin molecule.

In addition, the spectra performed at various incubation timesshowed that the interaction between -Lg and polyphenol was

detectable starting from 4 h of incubation. At that time, the relativeabundance of the -Lg/polyphenol adducts was still much lowerthan that of the native proteins. The abundance of the adducts in-creased with the incubation time. These findings also excluded thatthe ions observed were due to cluster artifacts of MALDI analysis.

The stable nature of the -Lg/polyphenol adducts was supported bytheir persistence in MALDI TOF MS following denaturing RP-HPLCseparation.

3.5. In vitro reactivity of catechin and epicatechin with -Lg

Once proved that -Lg was the only major milk protein to stablybind cocoa polyphenols, we investigated the nature of the protein bind-ing site.

a) Native -Lg (constituted by a mixture of the two genetic variants Aand B).

b) -Lg alkylated to block the single free cysteine at position 121, inorder to study its possible involvement in polyphenol binding.

c) Lactosylated -Lg obtained by RP-HPLC purification from total WPextracted from a sample of heat-treated milk. In this latter case, weaimed to define whether heat treatment employed to prepare com-mercial milk/chocolate beverages might change -Lg binding topolyphenols. In -Lg, it is known that the preferential site oflactosylation is located at lysine 100 (Fogliano et al., 1998).

Fig. 5 shows the MALDI-TOF-MS analysis of native -Lg (control inFig. 5a) incubated with catechin (Fig. 5b) and an epicatechin (Fig. 5c)for 24 h: both A and B variants added a single polyphenol molecule. Onthe contrary, -Lg treated with IAA, in which the free cysteine wasblocked, showed a molecular mass of 18,423 and 18,339 Da for variantsA and B, respectively, corresponding to the mass increase of 57 massunits for alkylation of the single free cysteine of the protein (Fig. 5df).However, upon incubation with polyphenols, no mass increase was

Fig. 2.ABTS assay of catechin, epicatechin,OPC andof thepolyphenols extracted from cocoa powder after incubationwith (a) casein, (b)whey proteins and (c)-Lg of cowmilk (values areexpressed asmillimoles of Trolox/liter);Mean (SD) from three separate experiments run in duplicate. *p b 0.05 versus t = 0; the ratio decrease (%)with respect to the initial time (t = 0)is indicated.


observed, indicating that the polyphenol binds specifically to the freecysteine residue.

In order to confirm this hypothesis, and to assign the reactive site of-Lg with polyphenols, tryptic hydrolysis was performed on thesamples after a diafiltration step to remove polyphenol excess fromthe reaction mixture. Fig. 6b shows the partial MALDI spectrum of thetryptic digest of native -Lg before and after incubation with catechin(control in Fig. 6a). The identification of peptides was performed onthe basis of the measured peptide masses compared with thoseexpected on the basis of trypsin specificity (Table 1) using the dataprocessing software GPMAW (Applied Biosystems).

Themass spectra obtained after incubation of epicatechin were verysimilar to that obtained for catechin incubation, demonstrating thesame extent of reactivity of the two phenols with -Lg. These data arein agreement with literature data that demonstrate two flavanolsexhibit the same behavior in terms of reactivity, chemical properties,etc. (Mendoza-Wilson & Glossman-Mitnik, 2006).

Peptide mass mapping allowed to verify the protein sequencecompletely. In particular, signals at m/z 2647.09 and 2675.14corresponded to peptides 102124 of -Lg variants B and A respec-tively. These signals, in the -Lg sample incubated with catechin

(Fig. 6b), were accompanied by signals at m/z 2935.83 and 2963.89where the mass of the two peptides was increased by 288 unitscorresponding to addition of a single catechin monomer. A similar resultwas obtained for epicatechin. In the case of IAA--Lg (Fig. 6c), peptide102124 was present only in the alkylated form but no evidence of cate-chin binding was found. In fact, peptide 102124 contains three cysteineresidues, two of which involved in a disulfide bridge and the third onefree. This result supported the hypothesis that the polyphenol couldhave reactedwith the free cysteine residue on the peptide, and alkylationof this residue prevented interaction either flavanols. For unambiguousstructural characterization, peptides at m/z 2961.40 and m/z 2933.36were analyzed by ESI-Q-TOF MS/MS (not shown) confirming binding ofpolyphenols to cysteine 121. Moreover, analysis showed the absence ofthese peptides in the case of alkylated -Lg, confirming, also in thiscase, the data provided by MALDI-TOF-MS.

The same analysis was also carried out on the lactosylated formof -Lg. The tryptic map of Lac--Lg presented the same peptides102124 with addition of catechin (signals m/z 2933.83 and2961.89) (Fig. 6d). This demonstrated that lactosylation did notprevent polyphenol binding, and that lactosylated -Lg covalentlybound polyphenols at the same cysteine 121 residue.

image of Fig.2

Fig. 3.MALDI-TOFmass spectra ofwhey proteins after 0 (a), after 24 h (b) and48 h (c) incubationwith polyphenol extracts from cocoa, excluding formation of covalent complexes of-Lawith cocoa polyphenols. *: Adduct with the matrix; Lac: Lactose.


3.6. LC ESI MS analysis of polyphenol/-Lg adducts in commercial products

LCESI-MS analysis of the intact whey protein fraction isolated from acommercial pasteurized chocolate/milk did not evidence any adduct pos-sibly because of the low extent of modification (not shown). However,the same analysis carried out on the tryptic digest of the same fractionmonitoring the triply charged ions of reacted peptides (Fig. 7d) showedthe presence of the same peptides 102124 of -Lg with additionof catechin/epicatechin which had been observed in the samplesincubated in vitro (Fig. 7ac) although to a very low extent. Thesefindings proved that covalent interactions -Lg/polyphenol occuralso in real milk and milk/chocolate beverages and adduct formationcan be monitored using the proposed peptidomic approach, once arobust quantification method has been developed.

4. Discussion

Several studies have highlighted the nutritional implications ofinteractions of polyphenolswith proteins in food, whichmay determinea decreased bioavailability of polyphenols. The loss of food nutritional

Fig. 4.MALDI-TOFmass spectra showing evidence of a complex between -Lg and (b) polyphencontrol (a).

quality has been ascribed tomodification of essential amino acid res-idues and to reduction of protein digestibility due to inhibition ofproteolytic and glycolytic enzymes. According to some studies, poly-phenols may interact irreversibly with dietary proteins and digestiveenzymes in the gut and be transported in vivo bound to plasmaproteins (Brunet, Blade, Salvado, & Arola, 2002). This protein bindingcan affect the physiological effects of polyphenols, depending ontheir intake and structure. Indeed, binding to food proteins mayhave implications in terms of bioavailability (Serafini, Ghiselli, &Ferro-Luzzi, 1996; Wollgast & Anklam, 2000), as the antioxidantcapacity of polyphenols can be modified by the presence of proteins(Arts, Haenen, Voss, & Bast, 2001; Arts et al., 2002; Riedl &Hagerman, 2001). Therefore, the proteins present in the food matrix,the digestive environment and the blood have the potential to signif-icantly affect the biological activity of polyphenols.

Despite this, the structural definition of the nature of these proteinpolyphenol complexes has never been carried out. Several kinds ofinteractions have been hypothesized: strong (covalent, ionic) or weak(hydrogen bridges, bonds, hydrophobic) bonds. With regard to thecovalent adducts, there are few data in the literature to support

ols extracted from cocoa, (c) catechin, (d) epicatechin, after 24 h incubation, compared to

image of Fig.3image of Fig.4

Fig. 5. MALDI-TOF-MS analysis of -Lg: a) control; b) after 24 h incubation with epicatechin; c) after 24 h incubation with catechin. MALDI-TOF-MS analysis of IAA--Lg: d) control;e) after 24 h incubation with epicatechin, f) after 24 incubation with catechin. *: adduct of the protein with the sinapinic acid matrix.


experimentally their formation and to identify binding sites of proteins.This study demonstrates for the first time the formation of the com-plex proteinpolyphenol involving a milk protein, -Lg and cocoapolyphenols, through covalent binding of free-SH group of the freecysteine residue of the protein.

To this aim, a strategy combining proteomic with biochemical ap-proaches has been applied to the characterization of a simplified modelsystem and then of a commercial products, a chocolatemilk drink.

The polyphenol extract of commercial cocoa was characterized byLCESIMS/MS and incubated with milk proteins. The interaction andcomplex formation at several incubation times was studied by massspectrometry and antioxidant activity assays. Antioxidant activity ofpolyphenols was measured by ABTS in a model assay system, whichwas shown to preserve antioxidant activity of polyphenols under theconditions of time/temperature/medium used for the interaction study.

The measurement of antioxidant activity of casein/polyphenol mix-tures revealed a time-dependent decrease of activity. It has been reportedthat the interaction with polyphenols modifies casein conformationresulting in a reduction of -helices and -sheets (Hasni et al., 2011).This decrease of antioxidant activity was observed also for polyphenol/WP and polyphenol/-Lg systems. Considering that polyphenols incubat-ed in the absence of proteins did not change significantly their anti-oxidant activity it was possible to hypothesize that the reducedantioxidant activity of polyphenolprotein system is due to a stableproteinpolyphenol interaction, resulting in a decrease of freepolyphenol, with a decrease of the antioxidant activity of the system.

Proteomic analysis, however, confirmed the formation of covalentcomplexes only in the case of WP, not for caseins. More precisely,while -La presents no reactivity with polyphenol extracts of cocoa aswell as with phenol standards, -Lg showed a significant reactivity tocatechin and epicatechin experimentally measurable starting from 4 hof incubation and remained stable up to 48 h. Taken together, thesedata suggest that covalent binding occurswith-Lg,while otherweaker,likely non-covalent interactions take place in the case of casein withcocoa polyphenols.

MALDI TOFMS and ESI-Q-TOFMS/MS analyses of tryptic peptides of-Lg have allowed the identification of the binding site as the free thiolgroup of cysteine 121. In support of our findings, reactivity of grapeflavan-3-ols, including catechins, with cysteine and cysteine derivativeshas already been described (Tanaka, Kusano, & Kouno, 1998; Torres &Bobet, 2001; Torres, Lozano, & Maher, 2005; Torres et al., 2002). Inany case, this is the first structural report of a food polyphenol covalentadducts with milk proteins.

Interestingly, grape polyphenol adducts with cysteine thiol groupsshowed a higher antiradical capacity than their underivatized counter-parts (Torres et al., 2002), and this suggests that functional properties of-Lg/polyphenol adducts deserve further detailed investigation, possi-bly on pure compounds obtained by chemical synthesis.

At the same time, our data showed also that only a small part ofthe protein interacts with the polyphenol; therefore, it can be con-cluded that the implications of nutritional point of view are notsuch as to support the hypothesis of a quantitative decrease in the

image of Fig.5

Fig. 6. Partial MALDI-TOF-MS analysis of the tryptic digest of -Lg incubated for 4 h with polyphenols: (a) control, and (b) native -Lg, (c) alkylated -Lg, (d) lactosylated -Lgincubated with catechin (CAT).

Table 1Identification of tryptic peptides of native -Lg, Lac--Lg and IAA--Lg after 24 h of incubation with catechin (CAT) or epicatechin (EPI). CM: peptide carboxymethylated. x: peptidedetected.

Peptide Measured MH+ Theoretical MH+ Peptide sequence -Lg Lac--Lg IAA--Lg

7175 573.55 573.36 IIAEK x x x7883 674.19 674.42 IPAVFK x x x142148 837.55 837.48 ALPMHIR x x x8491 916.51 916.47 IDALNENK x x x18 933.65 933.54 LIVTQTMK x x x92100 1065.22 1065.58 VLVLDTDYK x x125138 1635.89 1635.77 TPEVDDEALEKFDK x x x149162 1658.85 1658.78 LSFNPTQLEEQCHI x x4160 2313.54 2313.26 VYVEELKPTPEGDLEILLQK x x x102124 B 2647.11 2647.20 YLLFCMENSAEPEQSLACQCLVR x x x102124 A 2675.42 2675.20 YLLFCMENSAEPEQSLVCQCLVR x x1540 2707.23 2707.38 VAGTWYSLAMAASDISLLDAQSAPLR x x x6169 B + 149162 2721.59 2721.111 WENGECAQK + LSFNPTQLEEQCHI x x x6169 A + 149162 2777.97 2779.11 WENDECAQK + LSFNPTQLEEQCHI x x6170 B + 149162 2848.70 28848.30 WENGECAQKK + LSFNPTQLEEQCHI x x x6170 A + 149162 2906.43 2906.30 WENDECAQKK + LSFNPTQLEEQCHI x x102124 B + CAT 2935.89 2935.83 YLLFCMENSAEPEQSLACQCLVR + CAT x x102124 A + CAT 2963.89 2963.44 YLLFCMENSAEPEQSLVCQCLVR + CAT x102124 B + EPI 2935.83 2935.42 YLLFCMENSAEPEQSLACQCLVR + EPI x x102124 A + EPI 2963.89 2963.40 YLLFCMENSAEPEQSLVCQCLVR + EPI x6170 A + 149162 + lactose 3173.29 3173.39 WENGECAQKK + LSFNPTQLEEQCHI + lactose x(102124B) CM 2704.44 2704.33 YLLFCMENSAEPEQSLACQCLVR x(102124A) CM 2704.37 2704.33 YLLFCMENSAEPEQSLVCQCLVR x


image of Fig.6

Fig. 7. LCESI-MS analysis in selected ionmonitoringmode of the tryptic digest of whey proteins incubatedwith catechin (a: total ion current); b: triply charged ion of 102124 from -Lgvariant B reactedwith CAT (979.0 m/z); c: triply charged ion of 102124 from-Lg variant A reactedwith CAT (988.3 m/z), compared to the tryptic digest of thewhey protein fraction of acommercial sterilized milk-chocolate beverage (d: monitoring of the modified 102124 peptide forms from both -Lg variants A and B).


bioavailability of polyphenols themselves. Furthermore, LC ESI MSanalysis of a commercial chocolate/milk drink demonstrated the for-mation of the same covalent complex also in this product.

5. Conclusions

A proteomic approach has been developed to monitor the forma-tion of stable polyphenol/protein complex in either laboratory orcommercial milk products. The results obtained allow us to hypoth-esize the use of this methodology for similar products like chocolatemilk drinks, chocolate milk, and formulated protein containingcocoa. The extension of this method to the quantitative level couldbe useful to understand the type of polyphenolprotein interactionin these products taking into account the characteristics of ingredi-ents and the technological parameters of the production process. Atthe same time, the observed protein effects on the antioxidantactivity decrease measured in vitro have allowed us to assume thepresence of other types of weak proteinpolyphenol interactionsthat reduce this activity. Currently, little is known about polyphenolabsorption, bioavailability, biodistribution, and metabolism, althoughthere is probably a common path. Consequently, further studies areneeded to identify the nature and strength of these interactions, inorder to assess the possible structural changes upon food ingestionand digestion As a matter of fact, the consequences of the interactionsin nutritional terms are not so easily measurable and generalizable asit should be taken into consideration a number of other factors, notleast the type of polyphenol and the nature of the protein matrix.

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The interaction of cocoa polyphenols with milk proteins studied by proteomic techniques1. Introduction2. Materials and methods2.1. Reagents and standards2.2. Phenol extraction, quantitation and characterization by LC/MS/MS2.3. Extraction of milk proteins2.4. RP-HPLC analysis of whey proteins2.5. Study of in vitro interaction of polyphenols with caseins and WP2.6. Alkylation and tryptic hydrolysis of -Lg2.7. MALDI-TOF-MS analysis2.8. ESIMS/MS analysis2.9. Antioxidant activity assays2.10. Statistical analysis

3. Results3.1. Analysis of phenolic components in cocoa by LC/ESIMS/MS3.2. Antioxidant activity of polyphenol compounds3.3. Total antioxidant activity of polyphenol/protein mixtures3.4. Characterization of protein/polyphenols interactions by MALDI-TOF-MS3.5. In vitro reactivity of catechin and epicatechin with -Lg3.6. LC ESI MS analysis of polyphenol/-Lg adducts in commercial products

4. Discussion5. ConclusionsReferences

Documents

The interaction of cocoa polyphenols with milk proteins studied by