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Food Research International 54 (2013) 406–415
Contents lists available at ScienceDirect
Food Research International
j ourna l homepage: www.e lsev ie r .com/ locate / foodres
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 Università 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 β-lactoglobulin (β-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 protein–polyphenol
interactions, probably weaker non-covalent bonds. From a
nutri-tional point of view, these data indicate that the β-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, Rémésy,
& Jiménez,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 (Sánchez-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, & Rémésy, 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-Raventós, Romero-Pérez,
Andrés-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)
406–415
& 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 protein–polyphenol in vitro interactions werestudied
by either MALDI-TOF or ESI mass spectrometry, showing amajor
reactivity of β-lactoglobulin (β-Lg) towards cocoa polyphenols.The
data acquired allowed us to characterize the molecular basis of
theinteraction between themain cocoa flavanols (catechin and
epicatechin)with β-Lg by MS/MS structural analysis. The effect of
non-enzymaticglycosylation, always occurring in commercial
milk–cocoa products dueto thermal treatments, on β-Lg interaction
with polyphenols wasstudied. These data were complemented by total
antioxidant capacityassays on the protein–polyphenol 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,β-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). α-Cyano-4-hydroxycinnamic acid(α-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-Haën (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 μm.
Total phenol concentration in the different extracts were
determinedspectrophotometrically by the Folin–Ciocalteu assay (Box,
1983) usinggallic acid as a standard. An aliquot of 125 μL of each
extract was mixedwith 125 μL of Folin–Ciocalteu 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 Å column (250 × 4.6 mm, particle size 5 μM)
(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: 20–30%
B (6 min), 30–40% B (10 min), 40–50% B (8 min), 50–90% B (8 min),
90–90% B(3 min), 90–20% 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 μL. 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 at−20 °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 μm;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 μL sample dissolved at a concentration
of 2 mg/mL in H2O/TFA 0.1%were injected. The chromatographic peaks
containing α-lactalbumin(α-La) and β-Lg were dried, freeze-dried
and finally stored in a freezerat−20 °C.
Lactosylated β-Lg (Lac-β-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
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408 M. Gallo et al. / Food Research International 54 (2013)
406–415
concentrations of 5.4 mg/mL, 5.7 mg/mL, and 2.9 mg/mL for
caseins,whey proteins and β-Lg, respectively. The cocoa polyphenol
extractand individual polyphenols were dissolved in methanol and
addedat a 10-fold molar excess compared to that of protein.
Protein–polyphenol mixtures were incubated at 37 °C and the
interactionmonitored for 0–48 h by antioxidant activity assays and
massspectrometry analysis.
2.6. Alkylation and tryptic hydrolysis of β-Lg
Alkylation of β-Lg (IAA-β-Lg). β-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 μL of
the protein sampleand using sinapinic acid (10 mg/mL in 50%
ACN/TFA0.1%) as matrix. Forpeptide analysis, 1 μL of peptide
mixture was loaded on the appropriatetarget steel using α-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 β-Lg using General
Protein/MassAnalysis for Windows software (GPMAW).
2.8. ESI–MS/MS analysis
ESI–MS/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 β-Lgtryptic peptides
was carried out using a C18 reversed-phase column(218TP52, 5 μm,
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 5–70% 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 Tukey–Kramer 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/ESI–MS/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 LC–MS/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 β-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 orβ-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
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Fig. 1. (a) ABTS assay for standards and polyphenol extracts
from cocoa powder (values are expressed asmillimoles of Trolox/L).
(b) Folin–Ciocalteu 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.
409M. Gallo et al. / Food Research International 54 (2013)
406–415
reduction ofα-helix and β-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. 2b–c shows the results of ABTS assay after incubation of
individualphenols with whey proteins and with purified β-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 β-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 protein–polyphenol 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 α-La with extracts of cocoa polyphenols, catechin
orepicatechin over 24–48 h incubation time (Fig. 3). The same
spectra,instead, showed a significant reactivity for β-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 β-Lg and polyphenol
was
detectable starting from 4 h of incubation. At that time, the
relativeabundance of the β-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 β-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
β-Lg
Once proved that β-Lg was the only major milk protein to
stablybind cocoa polyphenols, we investigated the nature of the
protein bind-ing site.
a) Native β-Lg (constituted by a mixture of the two genetic
variants Aand B).
b) β-Lg alkylated to block the single free cysteine at position
121, inorder to study its possible involvement in polyphenol
binding.
c) Lactosylated β-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 β-Lg binding
topolyphenols. In β-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 β-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, β-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. 5d–f).However, upon incubation with
polyphenols, no mass increase was
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Fig. 2.ABTS assay of catechin, epicatechin,OPC andof
thepolyphenols extracted from cocoa powder after incubationwith (a)
casein, (b)whey proteins and (c)β-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.
410 M. Gallo et al. / Food Research International 54 (2013)
406–415
observed, indicating that the polyphenol binds specifically to
the freecysteine residue.
In order to confirm this hypothesis, and to assign the reactive
site ofβ-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 β-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 β-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 102–124 of β-Lg variants B and A
respec-tively. These signals, in the β-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-β-Lg
(Fig. 6c), peptide102–124 was present only in the alkylated form
but no evidence of cate-chin binding was found. In fact, peptide
102–124 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 β-Lg, confirming,
also in thiscase, the data provided by MALDI-TOF-MS.
The same analysis was also carried out on the lactosylated
formof β-Lg. The tryptic map of Lac-β-Lg presented the same
peptides102–124 with addition of catechin (signals m/z 2933.83
and2961.89) (Fig. 6d). This demonstrated that lactosylation did
notprevent polyphenol binding, and that lactosylated β-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 ofα-Lawith cocoa
polyphenols. *: Adduct with the matrix; Lac: Lactose.
411M. Gallo et al. / Food Research International 54 (2013)
406–415
3.6. LC ESI MS analysis of polyphenol/β-Lg adducts in commercial
products
LC–ESI-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 102–124
of β-Lg with additionof catechin/epicatechin which had been
observed in the samplesincubated in vitro (Fig. 7a–c) although to a
very low extent. Thesefindings proved that covalent interactions
β-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 β-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
protein–polyphenol complexes has never been carried out. Several
kinds ofinteractions have been hypothesized: strong (covalent,
ionic) or weak(hydrogen bridges, π 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 β-Lg: a) control; b) after 24 h
incubation with epicatechin; c) after 24 h incubation with
catechin. MALDI-TOF-MS analysis of IAA-β-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.
412 M. Gallo et al. / Food Research International 54 (2013)
406–415
experimentally their formation and to identify binding sites of
proteins.This study demonstrates for the first time the formation
of the com-plex protein–polyphenol involving a milk protein, β-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
chocolate–milk drink.
The polyphenol extract of commercial cocoa was characterized
byLC–ESI–MS/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 α-helices and β-sheets
(Hasni et al., 2011).This decrease of antioxidant activity was
observed also for polyphenol/WP and polyphenol/β-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
polyphenol–protein system is due to a stableprotein–polyphenol
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 α-La presents no reactivity with polyphenol
extracts of cocoa aswell as with phenol standards, β-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
occurswithβ-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
ofβ-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 ofβ-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
β-Lg incubated for 4 h with polyphenols: (a) control, and (b)
native β-Lg, (c) alkylated β-Lg, (d) lactosylated β-Lgincubated
with catechin (CAT).
Table 1Identification of tryptic peptides of native β-Lg,
Lac-β-Lg and IAA-β-Lg after 24 h of incubation with catechin (CAT)
or epicatechin (EPI). CM: peptide carboxymethylated. x:
peptidedetected.
Peptide Measured MH+ Theoretical MH+ Peptide sequence β-Lg
Lac-β-Lg IAA-β-Lg
71–75 573.55 573.36 IIAEK x x x78–83 674.19 674.42 IPAVFK x x
x142–148 837.55 837.48 ALPMHIR x x x84–91 916.51 916.47 IDALNENK x
x x1–8 933.65 933.54 LIVTQTMK x x x92–100 1065.22 1065.58 VLVLDTDYK
x x125–138 1635.89 1635.77 TPEVDDEALEKFDK x x x149–162 1658.85
1658.78 LSFNPTQLEEQCHI x x41–60 2313.54 2313.26
VYVEELKPTPEGDLEILLQK x x x102–124 B 2647.11 2647.20
YLLFCMENSAEPEQSLACQCLVR x x x102–124 A 2675.42 2675.20
YLLFCMENSAEPEQSLVCQCLVR x x15–40 2707.23 2707.38
VAGTWYSLAMAASDISLLDAQSAPLR x x x61–69 B + 149–162 2721.59 2721.111
WENGECAQK + LSFNPTQLEEQCHI x x x61–69 A + 149–162 2777.97 2779.11
WENDECAQK + LSFNPTQLEEQCHI x x61–70 B + 149–162 2848.70 28848.30
WENGECAQKK + LSFNPTQLEEQCHI x x x61–70 A + 149–162 2906.43 2906.30
WENDECAQKK + LSFNPTQLEEQCHI x x102–124 B + CAT 2935.89 2935.83
YLLFCMENSAEPEQSLACQCLVR + CAT x x102–124 A + CAT 2963.89 2963.44
YLLFCMENSAEPEQSLVCQCLVR + CAT x102–124 B + EPI 2935.83 2935.42
YLLFCMENSAEPEQSLACQCLVR + EPI x x102–124 A + EPI 2963.89 2963.40
YLLFCMENSAEPEQSLVCQCLVR + EPI x61–70 A + 149–162 + lactose 3173.29
3173.39 WENGECAQKK + LSFNPTQLEEQCHI + lactose x(102–124B) CM
2704.44 2704.33 YLLFCMENSAEPEQSLACQCLVR x(102–124A) CM 2704.37
2704.33 YLLFCMENSAEPEQSLVCQCLVR x
413M. Gallo et al. / Food Research International 54 (2013)
406–415
image of Fig.�6
-
Fig. 7. LC–ESI-MS analysis in selected ionmonitoringmode of the
tryptic digest of whey proteins incubatedwith catechin (a: total
ion current); b: triply charged ion of 102–124 from β-Lgvariant B
reactedwith CAT (979.0 m/z); c: triply charged ion of 102–124
fromβ-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
102–124 peptide forms from both β-Lg variants A and B).
414 M. Gallo et al. / Food Research International 54 (2013)
406–415
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 polyphenol–protein
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
protein–polyphenol 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 β-Lg2.7. MALDI-TOF-MS analysis2.8. ESI–MS/MS
analysis2.9. Antioxidant activity assays2.10. Statistical
analysis
3. Results3.1. Analysis of phenolic components in cocoa by
LC/ESI–MS/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 β-Lg3.6. LC ESI MS analysis of polyphenol/β-Lg adducts in
commercial products
4. Discussion5. ConclusionsReferences