Carbohydrate Polymers 236 (2020) 116044

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

The effect of pectic polysaccharides from grape skins on salivary protein – T

procyanidin interactions
Elsa Brandãoa,1, Ana Fernandesa,1, Carlos Guerreiroa, Manuel A. Coimbrab, Nuno Mateusa,
Victor de Freitasa, Susana Soaresa,*
a
REQUIMTE/LAQV - Laboratório Associado para a Química Verde, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo
Alegre, 687, 4169-007 Porto, Portugal
b
QOPNA & REQUIMTE\LAQV - Laboratório Associado para a Química Verde, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, water and chelator-soluble pectic polysaccharide fractions were obtained from white grape skins,
Astringency aiming to study their impact on the interaction between low polymerized grape seed procyanidins and salivary
Pectin proteins. Water and chelator-soluble polysaccharide fractions were composed by uronic acids and neutral sugars,
Procyanidins mainly arabinose and galactose, with water polysaccharide fraction showing a higher amount of branched pectic
Salivary proteins
polysaccharides. Both polysaccharide fractions were able to mitigate salivary protein-procyanidin interactions,
by a competition mechanism, resulting in a decrease of the amount of precipitated protein. Water polysaccharide
fraction was the most effective in inhibiting salivary protein precipitation, especially for acidic proline-rich
proteins, due to the higher affinity to interact with procyanidins (KA = 22222 M−1 and KA = 365 M-1 for water
and chelator polysaccharides, respectively). The interaction between polysaccharides and procyanidins showed
to be mainly governed by hydrophobic effect.

1. Introduction PC (tannins) and cell wall polysaccharides, although polysaccharides

Polyphenols associations with food macromolecules such as proteins
and polysaccharides are fundamental factors affecting the nutritional
and organoleptic attributes of polyphenol-rich food products such as
astringency, bitter taste, and color (Le Bourvellec & Renard, 2012). For
instance, astringency is generally accepted to be due to specific inter-
actions between salivary proteins (SP) and tannins. These interactions
can result in the formation of (in)soluble aggregates that precipitate,
causing a sensation of roughness, dryness, and constriction due to the
lack of lubrication (Baxter, Lilley, Haslam, & Williamson, 1997; Ma
et al., 2014; Soares, Brandão, Mateus, & de Freitas, 2017). Polyphenol-
polysaccharide interactions have also been shown to occur, affecting
significantly polyphenols extractability and their functional and nutri-
tional properties (Renard, Watrelot, & Le Bourvellec, 2017; Zhu, 2018).
SP-procyanidin (PC) interactions have been extensively studied but
much less information is available regarding the interactions between
have been shown to influence tannin-protein aggregation, possibly
leading to a change in astringency perception (Riou, Vernhet, Doco, &
Moutounet, 2002; Soares, Mateus, & de Freitas, 2012; Watrelot, Schulz,
& Kennedy, 2017).
PC are polyphenols which belong to the class of condensed tannins
and may differ by their degree of polymerization and by the type of
interflavanic linkage (Cheynier, 2005). Flavan-3-ol units are most fre-
quently linked via B-type bonds, that is, C4-C8 and C4-C6 and they can
also be esterified with gallic acid. Pectic polysaccharides, major con-
stituents of plant cell walls, have been demonstrated to bind strongly to
polyphenols in solution, particularly PC and anthocyanins (Padayachee
et al., 2012; Watrelot, Le Bourvellec, Imberty, & Renard, 2013). These
associations are fast and spontaneous, usually involving weak non-
covalent interactions such as hydrogen bonds and electrostatic and
hydrophobic interactions (Padayachee et al., 2012). However, pectic
polysaccharides are very heterogeneous plant polymers, with different

Abbreviations: AIR, alcohol-insoluble residue; aPRPs, acidic proline-rich proteins; bPRPs, basic proline-rich proteins; CSP, chelator-soluble pectic polysaccharides;
ECG, epicatechin gallate; gPRPs, glycosylated proline-rich proteins; GD, galloylated dimer; PC, procyanidins; PRPs, proline-rich proteins; SP, salivary proteins; TFA,
trifluoroacetic acid; TS, total sugar content; UA, uronic acids; WSP, water-soluble pectic polysaccharides
⁎
Corresponding author.
E-mail addresses: elsa.brandao@fc.up.pt (E. Brandão), ana.fernandes@up.pt (A. Fernandes), up201911054@fc.up.pt (C. Guerreiro), mac@ua.pt (M.A. Coimbra),
nbmateus@fc.up.pt (N. Mateus), vfreitas@fc.up.pt (V. de Freitas), susana.soares@fc.up.pt (S. Soares).
1
These authors contributed equally to this work

https://doi.org/10.1016/j.carbpol.2020.116044
Received 2 October 2019; Received in revised form 31 January 2020; Accepted 19 February 2020
Available online 20 February 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
E. Brandão, et al.
structural domains, which may affect their interaction with polyphenols
(Watrelot et al., 2017).
Pectic polysaccharides are predominantly constituted by α1→4
linked D-galacturonic acid residues (α-D-GalpA) that can be methyl es-
terified and/or acetylated, forming “smooth regions” of homo-
galacturonnans. The branched regions correspond to rhamnogalactur-
onans (RG). Type I RG are formed by repeating units of →4)-α-D-GalpA-
(1→2)-α-L-Rhap-(1→), with the rhamnose residues substituted by long
side-chains composed mostly of Gal and Ara residues. RG II present a
backbone of (1→4) linked α-D-GalpA residues with side chains con-
taining rhamnose and a variety of monosaccharide residues (Ochoa-
Villarreal, Aispuro-Hernández, Vargas-Arispuro, & Martínez-Téllez,
2012; Perez, Mazeau, & du Penhoat, 2000). The affinity of pectin to PC
depends on factors like structure, composition, and concentration of
both polysaccharides and PC, which can modulate the interactions
between these biomolecules.
Therefore, the aim of this work was to obtain new insights about the
effect of pectic polysaccharides fractions isolated from grape skins on
low-polymerized PC-SP interaction. For this purpose, pectic poly-
saccharides fractions were obtained by sequential extraction with water
(WSP) and chelator (CSP) solutions. Their effect on SP-PC interactions
was then evaluated by HPLC and SDS-PAGE. The binding affinity be-
tween PC and polysaccharide fractions was assessed by isothermal ti-
tration calorimetry (ITC).
2. Materials and methods
2.1. Plant materials
White grapes (Vitis vinifera L.) at maturity, kindly provided by
Lavradores de Feitoria®, were collected from a vineyard located in Cima
Corgo in the Douro Region. Grapes were brought to the laboratory and
frozen at -20 °C. Skins were separated from seeds and pulp, frozen and
freeze-dried until further analysis.
2.2. Isolation of PC
PC were obtained from a commercial grape seed extract (Vitis vi-
nifera L.) purchased to Vitisol®– berkem. This extract was mainly
composed by oligomeric PC with different degrees of polymerization
and 2 g were fractionated in a TSK-Toyopearl HW-40(s) gel column
(100 mm x 10 mm i.d.), at 0.8 mL.min−1, yielding five fractions as
described in the literature (de Freitas, Glories, Bourgeois, & Vitry,
1998).
The first three fractions were obtained after elution with 99.8 % (v/
v) methanol during 15 min, other 15 min and 4 h, respectively. The last
two fractions were eluted with methanol/5% (v/v) acetic acid during
the next 14 h and next 8 h, respectively. Deionized water was added to
these fractions, and the organic solvent was eliminated in a rotary
evaporator under reduced pressure at 30 °C and then freeze-dried.
PC composition of each fraction was determined by LC–MS by
Electrospray Ionization-Mass Spectrometry (ESI-MS) (Finnigan DECA
XP PLUS). Fraction containing PC dimers and galloyl derivatives was
chosen to perform the interaction studies.
2.3. Isolation of human saliva
Saliva was collected from several volunteers (healthy and non-
smoking) and treated as previously described (Brandao, Soares, Mateus,
& de Freitas, 2014). Briefly, 900 μL of saliva was acidified with 25 μL of
trifluoroacetic acid (TFA, final concentration of 0.1 %). The solution
was then centrifuged at 5914 g for 5 min and the supernatant was se-
parated from the precipitate. Saliva collection was conducted according
to the Declaration of Helsinki and was approved by the Ethics Com-
mittee of Medical School of University of Porto (EK84032011).
2
Carbohydrate Polymers 236 (2020) 116044
2.4. Isolation of pectic polysaccharides
An alcohol-insoluble residue (AIR) was obtained from white grape
skins according to an adaptation of the methodology described in the
literature (Hernandez-Hierro et al., 2014). Grounded grape skins were
suspended in boiling water for 5 min and homogenized using an Ultra-
Turrax® for 2 min. Absolute ethanol was added (70 % ethanol final
concentration) and the mixture was placed into an ultrasound bath for
15 min at room temperature and then into a water bath for 30 min at 40
°C, under magnetic stirring. The alcohol insoluble material was sepa-
rated by centrifugation (2862 g, 15 min, room temperature) and ex-
tracted again using 70 % ethanol solution. The washing procedure with
fresh 70 % ethanol was repeated several times until a clear extract was
obtained. Finally, the AIR was washed twice with 96 % ethanol, once
with acetone and dried overnight in an oven at 45 °C.
Consecutive fractional extraction of the AIR was performed as fol-
lows, according to the methodologies described by Slavov et al. (Slavov,
Panchev, Kovacheva, & Vasileva, 2016, 2017): 1) Hot-water extraction:
AIR (7.5 g) was extracted with hot water (150 mL distilled water) at 90
°C for 1 h with constant stirring. The residue was separated by cen-
trifugation (2862 g, 15 min, room temperature) and subjected to an-
other extraction with hot water. Then, the filtrates were combined and
evaporated under vacuum to 1/3 of their initial volume. Three volumes
of absolute ethanol were added to the concentrated liquid containing
the polysaccharides and left for 24 h at 4 °C. In order to completely
remove the ethanol, the precipitate obtained was centrifuged (2862 g,
15 min), re-dissolved in water and evaporated. Then, the concentrated
solution was dialysed (Spectra/Por®, 6−8 kDa cut-off) against distilled
water (1:100, sample:distilled water) with water changes at each 2 h at
the first 8 h and then at each 8 h till 48 h, freeze dried and denoted as
water-soluble pectic polysaccharides (WSP); 2) Ammonium oxalate
extraction: The residue resultant from the hot water extraction was
treated with 150 mL of 50 mM oxalate solution at 50 °C for 1 h (pH 5)
with constant stirring. After centrifugation (2862 g, 15 min, room
temperature), the residue was subjected to a second extraction. Both
filtrates were combined and evaporated under vacuum to 1/3 of their
initial volume. Then, the concentrated filtrate was treated with ethanol
as described above and named as chelator-soluble pectic poly-
saccharides (CSP).
2.5. The influence of pectic polysaccharides on salivary protein-PC
interaction
2.5.1. HPLC analysis
SP and PC were analyzed by HPLC before and after SP interaction
with PC and pectic polysaccharides addition. A mixture of saliva (110
μL) and distilled water (70 μL) was used as the control condition. For PC
experiments in the absence of polysaccharides, a mixture of saliva (110
μL) and PC (3.0 g. L−1, final volume of 180 μL) was shaken, kept in-
teracting for 10 min and centrifuged (4930 g, 5 min). After cen-
trifugation, the SP and PC present in the supernatant were analyzed by
HPLC. For the ternary system involving SP, PC and polysaccharides, PC
firstly interacted with increasing pectic polysaccharides concentrations
(0.5, 0.8, 1.2 and 1.5 g.L−1) for 30 min. After centrifugation (8150 g, 5
min), saliva (110 μL) was added to the supernatant and kept interacting
for more 10 min. The resulting solutions were centrifuged (4930 g, 5
min), and 100 μL of the supernatant was analyzed by HPLC Lachrom
system (Merck Hitachi, L-7100) equipped with the D-7000 HSM soft-
ware. The analysis was performed at 25 °C with a Vydac C8 column
(150 × 2.1 mm; 5 μm) at 214 nm, using a UV–vis detector (L-7420).
The HPLC solvents were 0.2 % aqueous TFA (eluent A) and 0.2 % TFA
in ACN/water 80/20 (v/v) (eluent B). The initial conditions (15 %
eluent B) were maintained for 10 min, and after that, the gradient ap-
plied was linear from 15 to 60 % (eluent B) in 40 min, at a flow rate of
0.5 mL.min−1. The column was washed with 100 % eluent B for 10
min. After this washing, the column was stabilized with the initial
E. Brandão, et al.
conditions during 20 min.
PC were analyzed by HPLC/ESI-MS at 280 nm on an Agilent
Poroshell 120 EC-C18 column (150 × 4.6 mm i.d., 2.7 μm), according
to the literature (Garcia-Estevez, Alcalde-Eon, & Escribano-Bailon,
2017). The HPLC solvents were 0.5 % aqueous formic acid (eluent A)
and 0.5 % formic acid in ACN (eluent B). The gradient applied was
linear from 0 to 40 % (eluent B) in 55 min, at a flow rate of 0.5
mL·min−1. After the program, the column was washed with 100 %
eluent B to elute other PC and aggregates. After washing, the column
was stabilized with the initial conditions. MS analysis was done on a
Finnigan Surveyor series liquid chromatograph equipped with a Fin-
nigan Surveyor PDA Plus detector. Detection was carried out between
200 and 700 nm. The mass detection was carried out by a Finnigan LCQ
DECA XP MAX (Finnigan Corp., San José, Calif., USA) mass detector
with an API (Atmospheric Pressure Ionization) source of ionization and
an ESI interface. The capillary voltage was 4 V and the capillary tem-
perature 325 °C. Spectra were recorded in negative ion mode between
m/z 0 and 2000 with series of three scans: a full mass, a zoom scan of
the most intense ion in the first scan, and a MS/MS of the most intense
ion using relative collision energies of 30 and 60 V.
2.5.2. SDS-PAGE
The precipitates resulting from the interaction between SP and PC in
the absence or presence of pectic polysaccharide fractions (WSP and
CSP), as well as the saliva control, were analysed by SDS-PAGE fol-
lowing the method of Schägger (Schägger & von Jagow, 1987). This
method is based on a separation of proteins according to their sizes
using a 16 % acrylamide resolving gel. The precipitates were re-
solubilized in 50 μL of electrophoresis buffer (125 mM Tris−HCl pH
6.8, 20 % v/v glycerol, 4 % SDS, 10 % v/v β-Mercaptoethanol, and
0.004 % bromophenol blue) and heated at 90 °C for 20 min. The run-
ning buffer was 0.2 M Tris−HCl pH 8.3, 1.9 M glycine and 0.1 % SDS.
Molecular weight markers were broad ranged (Precision Plues Pro-
teinTM Unstained Standards, Bio-Rad). The separation was performed
on a C.B.S. Scientific Large Cell electrophoresis apparatus at constant
amperage (0.03 Å). After electrophoresis, the gels were stained with
Imperial Protein Stain - a Coomassie R-250 dye-based reagent - for 30
min. The destaining step was done by washing the gels overnight with
water:methanol:acetic acid (70:20:10 v/v/v).
2.5.3. Isothermal titration calorimetry (ITC)
To determine the enthalpy changes associated with pectic poly-
saccharides and PC interactions at 298 K, a V-P MicroCalorimeter
controlled by Origin VPViewer software was used. Aqueous solutions of
pectic polysaccharide fractions (5.8 mM and 6.8 mM galacturonic acid
equivalents, WSP and CSP, respectively) and of PC (titrant, 30 and 35
mM catechin equivalents) were prepared and degassed before mea-
surements. The polysaccharides solution was loaded into the sample
cell (1.4 mL) and the injection syringe was loaded with the titrant. After
the stabilization of the baseline, PC solution was injected (approxi-
mately 13–15 injections, 4–12 μL/injection) into the sample cell until
reaching stabilization. Spacing between injections was equal or higher
than 30 min. Samples were stirred constantly at 307 rpm to ensure
thorough mixing. Control experiments comprise the titration of single
PC fractions in milliQ water.
Raw data obtained as a plot of heat flow (microcalories per second)
against time (minutes) were integrated peak-by-peak and normalized to
achieve a plot of observed enthalpy change per mole of injectant (ΔH,
kcal. mol−1) against the molar ratio (PC/pectic polysaccharides).
NITPIC software was used to baseline establishment and peak integra-
tion, while the experimental data was fitted to a theoretical titration
curve using SEDPHAT software (Keller et al., 2012; Scheuermann &
Brautigam, 2015) with ΔH (enthalpy change) and Ka (association con-
stant) as adjustable parameters. Thermograms were done using GUSSI
software (Brautigam, 2015).
3
Carbohydrate Polymers 236 (2020) 116044
2.6. Analytical methods
2.6.1. Total polyphenols determination
The total polyphenolic content of each pectic polysaccharide frac-
tion was determined with the Folin-Ciocalteu assay. Pectic poly-
saccharide fractions were dissolved in distilled water (1.0 mg.mL−1)
and an aliquot of these solutions was mixed with Folin-Ciocalteu re-
agent, water and sodium carbonate solution (20 %) (Singleton & Rossi,
1965). After 30 min incubation at room temperature, absorbance was
measured at 750 nm in an UV/Vis spectrophotometer (PowerwaveXS
Microplate Reader). Gallic acid was used as standard and the results
were expressed as μg gallic acid equivalents per mg dry weight in ex-
tracts. The measurements were performed in triplicate.
2.6.2. Soluble proteins determination
Soluble protein content of each pectic polysaccharide fraction was
determined by a dye binding assay according to Lin et.al (Lin, Fischer, &
Wicker, 2016). Briefly, pectic polysaccharide fractions were dissolved
in distilled water (1.0 mg.mL−1) and an aliquot of these solutions was
mixed with Bradford reagent in a 96 well microplate, incubated for 10
min at room temperature, and absorbance was measured at 595 nm.
Protein content in WSP and CSP was calculated relative to bovine serum
albumin (BSA) standard and was expressed as μg bovine serum albumin
equivalents (BSAE) per mg of fractions dry weight. The measurements
were performed in triplicate.
2.6.3. Polysaccharide analysis
Characterization of pectic polysaccharides was performed following
the procedure described by Nunes et al. (Nunes, Saraiva, & Coimbra,
2008). Monosaccharides were released from cell wall polysaccharides
by a pre-hydrolysis in 200 μL of H2SO4 (72 %) for 3 h at room tem-
perature, followed by 2.5 h hydrolysis in 1 M H2SO4 at 100 °C. Neutral
sugars composition was determined after conversion to their alditol
acetates by Gas Chromatography (GC), using 2-deoxyglucose as internal
standard. Results were expressed as μg sugar.mg−1 dry sample.
Uronic acids (UA) determination in the cell wall material were de-
termined by a modification of the 3-phenylphenol colorimetric method
(Blumenkrantz & Asboe-Hansen, 1973). Samples were prepared by
hydrolysis in 200 μL of H2SO4 (72 %) for 3 h at room temperature
followed by 1 h in 1 M H2SO4 at 100 °C. A calibration curve was made
with D-galacturonic acid. The hydrolysis of all samples was done in
duplicate and each one was analyzed twice. UA content was expressed
as μg galacturonic acid equivalent per mg dry sample. The degree of
methyl esterification of pectic polysaccharides was determined based
on the methanol content released after saponification (Nunes, Rocha,
Saraiva, & Coimbra, 2006; Waldron & Selvendran, 1990). Methanol was
quantified by gas chromatography with flame ionization detector (GC-
FID), using 1-propanol as internal standard.
2.7. Statistical analysis
The effect of polysaccharides on SP-PC interactions were performed
in triplicates (HPLC-DAD analysis) and expressed as mean values and
standard error of mean (SEM). Statistical significance was evaluated by
analysis of variance (ANOVA), followed by the Bonferroni’s test;
Differences were considered to be statistically significant at P < 0.05.
Concerning sugar analysis, the neutral sugars were analyzed in dupli-
cate, while the uronic acids were analyzed in triplicate and expressed as
mean values and standard deviations. All statistical data were processed
using GraphPad Prism version 5.0 for Windows.
3. Results and discussion
This work aimed to provide new information regarding the relative
affinity of dimeric PC and galloyl derivatives toward SP, together with
the impact of pectic polysaccharides (WSP and CSP) from white grape
E. Brandão, et al.
skins on these interactions. This information was achieved by three
complementary methodologies: HPLC analysis of SP and PC in the ab-
sence and in the presence of pectic polysaccharides, SDS-PAGE analysis
of precipitated SP, and ITC to evaluate the affinity of PC to interact with
pectic polysaccharides.
3.1. Composition of the interaction species
3.1.1. Pectic polysaccharides fractions
In order to clarify the binding between PC and pectic poly-
saccharides obtained from white grape skin, two pectic polysaccharide
fractions were obtained and characterized. The raw plant material was
initially treated with 70 % ethanol (40 °C), according to an adaption of
the De Vries method, in order to remove the low-molecular substances,
such as polyphenols, sugars, and salts, while obtaining the AIR. This
method has been described as appropriate for isolation of grapes cell-
wall material in comparison to other standard procedures (Apolinar-
Valiente, Romero-Cascales, Lopez-Roca, Gomez-Plaza, & Ros-Garcia,
2010). The AIR accounted for 17 % of the freeze-dried skin tissue (w/
w).
To obtain different polysaccharide fractions, the AIR was sequen-
tially extracted with water (WSP) and ammonium oxalate solution
(CSP). While pectic polysaccharides with a high degree of esterification
are solubilized by hot water, middle lamella pectic polysaccharides,
which are associated by calcium bridges, can be extracted by chelating
agents of calcium ions (Fugel, Carle, & Schieber, 2004). The two
polysaccharide fractions were obtained with lower yields (4.1 % and
2.3 %) (Table 1). Although lower than the 13 % obtained in a previous
study where HEPES and buffer-soluble polysaccharides were extracted
from skin tissues (Vidal, Williams, O’Neill, & Pellerin, 2001), these
values were in accordance with a less advanced stage of maturity of the
fruits used, as well as the use of ethanol, which forms a matrix that
prevents the extraction of polysaccharides. Compositional analysis of
the water and chelator fractions showed that their total sugar content
(TS) was very similar, consisting respectively of 77 % and 85 % by
weight of polysaccharides. Although polysaccharides were the main
constituents of the cell-wall material, soluble proteins and polyphenolic
compounds were also found in these fractions. Table 1 shows that the
polyphenolic content of these fractions corresponds to approximately 8
and 5% dry weight (WSP and CSP, respectively), while soluble protein
content was shown to be less than 4% for both fractions.
Carbohydrate analysis indicated that both fractions were char-
acterized by high UA content (709 and 820 μg. mg−1, on a dry weight
basis) and very low neutral sugars content, corresponding to 68 and 29
μg. mg−1. This indicates that these two fractions were rich in homo-
galacturonans (Watrelot et al., 2013). In fact, it is well known that
polysaccharides extracted by chelate extractants are mainly composed
by homogalacturonans associated intermolecularly through calcium
bridges, which form the so called “egg-box” structural features in plants
cell walls (Voragen, Coenen, Verhoef, & Schols, 2009). The degradation
of these complexes was accompanied by the release of part of the
homogalacturonans, resulting in higher UA content. However, ac-
cording to other data published in the literature, the water-soluble
Table 1
Chemical composition of the pectic polysaccharide fractions obtained by se-
quential extraction with water (WSP) and chelator (CSP) solutions. Results
expressed as average ± standard deviation (μg/mg dry matter).
Yield* (%) TS NS SP P these conditions. No information could be obtained regarding bPRPs,
(μg. mg−1) (μg. mg−1) (μg. mg−1) (μg. mg−1)
WSP 4.1 777 ± 136a 74 ± 12a 35.1 ± 0.2a 84 ± 4a
CSP 2.3 848 ± 42a 31 ± 3b 31 ± 1b 49 ± 1b
TS, total sugars; NS, neutral sugars; SP, soluble proteins; P, polyphenols. In the
same column, samples with different letters are significantly different
(p < 0.05).
4
Carbohydrate Polymers 236 (2020) 116044
fraction was expected to be comprised of higher amounts neutral sugars
(ca. 60–72 % Ara and Gal) (Vicens et al., 2009). Our results seem to
indicate that probably some of the skin water soluble polysaccharides,
particularly those containing Ara and Gal were not extracted under the
conditions used, which is a characteristic of unripe tissues (Nunes et al.,
2008). Main neutral sugars present in WSP were Ara (31 μg. mg−1) and
Gal (21 μg. mg−1), followed by a small amount of rhamnose (4 μg.
mg−1). The CSP fraction showed a lower amount of neutral sugars
when compared to WSP fraction, with the most abundant being Ara,
followed by Gal (Tables 1 and 2). This ratio was higher for CSP fraction
(2.7) in comparison with the WSP fraction (1.5), which could indicate a
higher amount of Ara or polysaccharides rich in Ara arising from the
pectic polysaccharide framework of this fraction (Apolinar-Valiente
et al., 2015, 2014). On the other hand, the ratio of (Ara + Gal) to
rhamnose was calculated to estimate the relative importance of the
neutral side-chains to the RG backbone, since it is assumed that most of
the Ara and Gal are associated with pectic polysaccharide hairy regions.
This ratio was lower for CSP fraction (7.3) than in WSP fraction (13.0),
which could indicate that CSP fraction contains shorter hairy regions of
pectic polysaccharides (RG-like structures) (Apolinar-Valiente et al.,
2015, 2014; Ducasse, Williams, Meudec, Cheynier, & Doco, 2010). The
UA/(Ara + Gal) ratio was lower for WSP (13.6) when compared to CSP
(37.2). The relatively higher proportion of Ara and Gal, as well as the
presence of UA, suggests that WSP has a higher amount of pectic
polysaccharide neutral sugars when compared to CSP fraction (Nunes
et al., 2008). Both polysaccharide fractions showed a similar release of
methanol (31 μg/mg and 39 μg/mg for WSP and CSP, respectively),
which corresponds to a degree of methyl esterification of approximately
25 %.
3.1.2. PC
A low polymerized PC fraction obtained after extraction and pur-
ification from grape seeds was selected for these interaction studies.
This fraction was analyzed by HPLC/ESI-MS allowing the identification
of several dimeric PC (B1, B3, B6, B4, B2, B7, B5 - major compounds,
corresponding to about 90 % of total PC content at 280 nm (λmax)) and
galloyl derivatives – galloylated dimer (GD) and epicatechin gallate
(ECG). Small amounts of a trimeric PC could also be detected on this
fraction. PC identification was performed based on the retention time of
standards compounds and by LC–MS analysis (Fig. 1).
3.2. Salivary protein-PC interaction
The relative affinity of a dimeric PC fraction toward specific SP was
assessed by HPLC analysis, through the determination of both unbound
PC and non-complexed SP after PC-proteins interaction and consequent
precipitation. The percentage of complexes was determined by sub-
tracting the area of each SP family (or PC) obtained in the HPLC ana-
lysis from the respective area of the control sample (saliva without PC
or PC alone).
Preliminary experiments were made in order to determine the
preferential PC concentration that lead to a significant precipitation of
the SP families: glycosylated proline-rich proteins (gPRPs), acidic pro-
line-rich proteins (aPRPs), statherin/P-B peptide and cystatins. It can be
seen in Fig. 2 that the addition of 3.0 g.L−1 of PC fraction practically
depleted aPRPs (∼95 %) and statherin/P-B peptide (∼85 %), and re-
duced the amount of cystatins (∼30 %). gPRPs were only slightly af-
fected by PC, evidencing a ∼5% reduction with these compounds in
due to overlapping with PC chromatographic peaks (data not shown).
In previous works, the same relative affinity has already been observed
with pure compounds (condensed and hydrolysable tannins) showing
the higher affinity of aPRPs and P-B peptide (Brandao, Soares, Mateus,
& de Freitas, 2014; Silva et al., 2017; Soares, Mateus, & de Freitas,
2012).
In order to understand the specificity and the impact of tannin
E. Brandão, et al.
Table 2
Composition of the extracted polysaccharide fractions obtained by sequential extraction with water (WSP) and chelator (CSP) solutions. The results are expressed as
average ± standard deviation μg/mg dry matter).
Rha Ara Xyl
(μg. mg−1) (μg. mg−1) (μg. mg−1)
WSP 3.8 ± 0.7a 32 ± 5a 6 ± 2a
CSP 2.6 ± 0.1a 15.8 ± 0.4b 1.8 ± 0.2b
UA, uronic acids; Rha – Rhamnose; Ara – Ara; Xyl – Xylose; Man – Mannose; Gal – Gal; Glc – Glucose. In the same column, samples with different letters are
significantly different (p < 0.05).
*Yield is expressed in mg of dry weight material per g of AIR.
structure on SP-PC interaction, PC profile was also analyzed by HPLC
before and after saliva interaction (Fig. 3).
The HPLC profile of PC fraction after saliva interaction evidenced a
slight but significant decrease on PC contents (Fig. 3). Galloyl deriva-
tives, GD and ECG, evidenced a higher affinity to these SP families,
resulting on a more notorious reduction of the amount of unbound
galloyl derivatives. This could be observed particularly for GD (∼44 %
bound PC) and to a smaller extension for the galloylated monomeric PC,
ECG (∼14 %). Within dimeric PC, B4 showed the biggest decrease
(∼14 %) significantly different in comparison with B1 and B6 (Table
S1, Supplementary Material). On the other hand, the other dimeric PC
showed similar amounts of bound tannins after protein interaction
(∼4-8%). The only exception was for PC B1 which did not show a
significant decrease, evidencing a lower protein affinity. Trimeric C1
also showed a slight decrease on the amount of free compound, sug-
gesting a lower affinity for these SP. All together, these results seem to
indicate that PC structure affects their interaction with SP, particularly
the presence of galloyl moieties. In fact, according to literature, the
increase of the degree of galloylation of proanthocyanidins increases
their ability to precipitate proteins (de Freitas & Mateus, 2001; Sarni-
Manchado & Cheynier, 2002).
3.3. Effect of pectic polysaccharides on salivary protein-PC interactions
3.3.1. HPLC analysis
In order to understand the impact of pectic polysaccharides on SP-
PC interactions, polysaccharides and PC were mixed together and let to
interact prior to saliva addition. This experimental approach intended
Fig. 1. 4HPLC chromatogram detected a 280 nm of the mixture of PC used in this work and respective identification. B1 to B7 – dimeric PC; GD – Galloylated Dimer;
ECG - epicatechin gallate.
5
Carbohydrate Polymers 236 (2020) 116044
Man Gal Glc UA
(μg. mg−1) (μg. mg−1) (μg. mg−1) (μg. mg−1)
2.5 ± 0.6a 20.7 ± 0.8a 4 ± 1ª,c 709 ± 149a
0.45 ± 0.01b 6 ± 2b 1.5 ± 0.2b 820 ± 44a
to mimic the natural occurring polyphenol-cell wall polysaccharide
interaction during fruit processing and beverages production. PC and
polysaccharides fractions (WSP and CSP) were prepared at different
polysaccharide concentration (0.5; 0.8; 1.2 and 1.5 g.L−1) and left to
interact for 30 min. Only after pectic polysaccharide-PC interaction and
centrifugation, saliva was added and allowed to stand for 10 min.
Fig. 4 shows the variation of the chromatographic peaks area of the
major SP families with increasing concentrations of WSP and CSP, ex-
pressed relatively to the saliva control, where saliva without PC and
polysaccharides accounted for 100 %.
Both WSP and CSP fractions were able to reduce the interaction
between SP and PC, showing a clear recovery of proteins chromato-
graphic peaks, particularly those for aPRPs and statherin/P-B peptide.
These results evidence that probably a solubilization of proteins-PC
complexes or a competition interaction between SP and polysaccharides
for PC binding may occur.
In general, the presence of the polysaccharide fractions induced a
significant recovery of the SP chromatographic peaks, although this
recovery was not complete. Increasing polysaccharide concentration
resulted on a higher protein recovery, except for the higher poly-
saccharide concentration (1.5 g.L−1) which did not evidence significant
difference compared to the previous concentration (1.2 g.L−1). Indeed,
it seems that there is a kind of stabilization at the polysaccharide
concentration of 1.2 g.L−1. The only exception is for aPRPs, where it
was observed a recovery with the highest WSP concentration.
Concerning gPRPs, it was observed that they were not affected by the
addition of pectic polysaccharides and did not show a significant dif-
ference for the control sample with increasing polysaccharide
E. Brandão, et al.
Fig. 3. PC composition variation after interaction with SP determined by HPLC
at 280 nm. Values with different letters within each PC are significantly dif-
ferent (p < 0.05).
concentrations (for both WSP and CSP). A detailed analysis of the ef-
ficiency of each polysaccharide fraction seemed to indicate that it de-
pends both on the SP but also on the polysaccharide concentration. For
instance, WSP has more influence on aPRPs-PC interaction than on
other SP families, being the most efficient polysaccharide in preventing
aPRPs precipitation. In fact, for the same WSP and CSP concentration, a
highest recovery of aPRPs chromatographic peak area could be ob-
served for WSP (∼90 %). For CSP, despite increasing polysaccharide
concentration, protein recovery could only be achieved up to ∼55 %.
This seems to indicate that specific structural features of these poly-
saccharide fractions could be inducing different affinities and simulta-
neously a change on proteins recovery efficiency. Statherin/P-B peptide
were co-eluted under the chromatographic conditions tested wherein,
showing a decrease of approximately 86 % of the chromatographic area
when compared to control sample, probably due to the higher P-B
peptide affinity towards tannins, as already reported in previous works
(Silva et al., 2017; Soares et al., 2016). Regarding cystatins, addition of
WSP caused a significant recovery of their chromatographic peak area
compared to SP-PC interaction at 0.8 and 1.2 g.L−1. Higher
6
Carbohydrate Polymers 236 (2020) 116044
Fig. 2. Influence of PC fraction (3.0 g. L−1) on
interaction with several SP families (gPRPs,
aPRPs, statherin/P-B peptide and cystatins)
determined by HPLC at 214 nm. A) Part of
chromatogram of SP before and after interac-
tion with PC; B) Variation of the chromato-
graphic peaks area of the several SP studied in
presence of PC with different concentrations.
Values with different letters within each SP are
significantly different (p < 0.05).
polysaccharide concentration did not induce an increase on protein
recovery. Considering CSP, all concentrations tested evidenced a pro-
tein recovery, although there was no statistically significant difference
at the highest concentration tested. The inhibition of SP precipitation
by polysaccharides was already reported for commercial pectins (Soares
et al., 2012a). Soares and co-workers studied the effect of several
commercial carbohydrates on the interactions between SP and grape
seed PC. Among the several carbohydrates studied (e.g. arabic gum,
pectin and poligalacturonic acid), the authors observed that commer-
cial citrus pectin was the most effective carbohydrate in preventing
precipitation of SP by PC. Although in that study the concentrations
used were different, aPRPs and statherin were the SP families that
showed the highest recovery in the presence of pectin (Soares et al.,
2012a).
3.3.2. SDS-PAGE
Besides the HPLC analysis of the supernatant resultant from saliva
interaction with PC fraction in the absence and presence of both pectic
polysaccharides, the resulting precipitates were also analyzed by SDS-
PAGE aiming to verify the efficiency of the selected pectic poly-
saccharides fractions in inhibiting SP-PC interactions (Fig. 5).
The results obtained by SDS-PAGE analysis of the precipitates de-
monstrated that interaction with WSP and CSP decreases SP that pre-
cipitate due to PC interaction. Although SDS-PAGE do not present all SP
families, this methodology provides information about the general in-
teraction profile. The protein band where polysaccharides had a higher
effect corresponds to PRPs, with a band at ∼24 kDa (Delius, Medard,
Kuster, & Hoffmann, 2017; Soares et al., 2011). As the intensity of this
band decreased as the concentration of polysaccharides increased, it
can be concluded that these polysaccharides are more effective towards
PRPs-PC interactions. This is particularly evident for WSP fraction.
These results are in agreement with the HPLC results, where it was
observed the highest recovery for the chromatographic peaks area of
PRPs due to less SP precipitation. In addition, increasing polysaccharide
concentration seemed to induce a higher precipitation of the protein
band corresponding to α-amylase (∼56 kDa), which is evidenced by
the increase of band intensity.
3.4. Interaction between pectic polysaccharides and PC by ITC
ITC is a very useful technique which provides direct thermodynamic
information associated with macromolecules complexes formation. ITC
allows to measure the binding equilibrium directly by determining the
E. Brandão, et al.
Fig. 4. Influence of increasing pectic polysaccharide concentration (0.0-1.5 g.L−1) on SP interaction with PC fraction (3.0 g.L−1) determined by HPLC at 214 nm.
Values with different letters within each SP family are significantly different (p < 0.05).
Fig. 5. SDS-PAGE of saliva (SP) and of the precipitates that resulted from the
interaction between SP and PC in the absence (SP + PC) (a) and in the presence
of increasing concentrations of polysaccharide fractions (CSP and WSP): b) 0.5
g.L−1; c) 0.8 g.L−1; d) 1.2 g.L−1; e) 1.5 g.L−1. The molecular weight markers
were substituted by lines, and the molecular mass marked on the left side is
expressed in kDa. The gel was stained with Imperial Protein Stain, a Coomassie
R-250 dye-based reagent.
heat involved in the association (generated or lost) of a ligand with its
binding partner in a calorimeter cell held under isothermal conditions
(Le Bourvellec & Renard, 2012). Fig. 6 shows the thermograms of the
titration of pectic polysaccharides fractions (CSP and WSP) by PC
fraction. The enthalpy changes per mole of injectant (ΔH, kcal. mol−1)
were plotted against the molar ratio (PC/pectic polysaccharides) and
the resulting curve was fitted using SEDPHAT software.
The titration of CSP and WSP by PC showed thermograms
7
Carbohydrate Polymers 236 (2020) 116044
characterized by weak exothermic peaks (heat release = -0.4 and 1.0
μcal/s)), while the blank experiment (injections of PC in milliQ water)
yielded small endothermic peaks (data not shown). Thermodynamic
parameters obtained through the fitting are presented in Fig. 6 (KA, ΔH
and ΔS). The global Gibbs free energy (ΔG) is negative for both inter-
actions (-3.50 and -5.93 kcal mol−1, for CSP and WSP, respectively)
which is a requirement for a spontaneous biological interaction. The
association constant KA was 365 M-1 for CSP-PC interaction, while for
WSP-PC interaction KA was 22222 M−1 which means that WSP fraction
has a higher affinity to interact with dimeric PC than CSP fraction. This
could explain the higher effect of this polysaccharide fraction on SPePC
interaction, since less amount of PC is available to interact with SP.
Analysis of the thermodynamic contributions indicated a positive en-
tropy change for both interactions which means that they are en-
tropically driven interactions such as hydrophobic interactions (Frazier
et al., 2010; Poncet-Legrand, Gautier, Cheynier, & Imberty, 2007;
Watrelot, Le Bourvellec, Imberty, & Renard, 2014). Moreover, this
analysis also shows a very small enthalpy contribution related to the
exothermic interaction (ΔH = - 0.297 and - 0.016 cal/mol, CSP and
WSP respectively). The origin of entropy contribution can be due to the
establishment of hydrophobic contacts with PC aromatic groups, which
have been considered of first importance for entropy-driven interac-
tions of tannins with biomolecules (Poncet-Legrand et al., 2007).
Overall, the results suggest that these pectic polysaccharides were
able to disrupt SP-PC interactions. The underlying mechanism of this
effect seems to be the competition one, in which WSP and CSP compete
with SP for PC binding. ITC allowed not only to suggest the underlying
the mechanism as well as to establish the binding affinity between PC
and pectic polysaccharides fractions. The results obtained by ITC
showed that WSP had a higher affinity to interact with PC and conse-
quently, was the most efficient polysaccharide fraction to reduce SP-PC
interaction. These results agree with the ones obtained by HPLC and
SDS-PAGE.
It was already described that acidic polysaccharides are able to af-
fect these interactions (Boulet et al., 2016; Vidal et al., 2004). Both
pectic fractions have in general a high amount of UA, but the UA
content of WSP fraction was lower than the CSP fraction. Furthermore,
the results suggest that WSP fraction had a higher amount of branched
pectic polysaccharides, carrying more neutral side chains compared to
CSP, mainly composed by hydrophobic residues such as Ara and Gal.
E. Brandão, et al.
Although it was already reported that neutral side chains can limit in-
teractions with PC (Watrelot et al., 2014; Zhu, 2018), in this work, it
seems that they can favor these interactions. However, it is important to
take into account that in most of the previous studies the authors used
PC with higher degree of polymerization (n = 9–30), while herein it
was used a PC mixture mainly composed by dimers. Furthermore, it was
demonstrated in this work that both polysaccharide fractions interact
with PC by hydrophobic interactions in which those hydrophobic re-
sidues can be involved, explaining the higher efficiency observed for
WSP fraction. Indeed, it was also reported that polysaccharides can
encapsulate PC by hydrophobic interactions, mostly lower polymerized
PC, due to the cavity dimension of the polysaccharide (Bindon, Smith, &
Kennedy, 2010). Moreover, there are some studies which refer that
some pectic water soluble polysaccharides can inhibit protein-tannin
aggregation due to the formation of tannin-polysaccharides complexes
(Carvalho et al., 2006; Riou et al., 2002; Watrelot et al., 2017).
4. Conclusions
In the present work it was demonstrated the potential of WSP and
CSP polysaccharide fractions from non-conventional origins, such as
grape skins, for the reduction/inhibition of SP-PC interactions. Both
polysaccharide fractions showed to act by a competition mechanism, in
which polysaccharides compete with SP by PC binding. Polysaccharides
interaction with PC showed to be mainly governed by hydrophobic
effect. WSP fraction evidenced a higher efficiency to reduce SP pre-
cipitation compared to CSP, mainly for aPRPs and statherin/P-B pep-
tide. This inhibitory effect could possibly be due to the presence of a
higher amount of neutral side chains composed mainly by Ara and Gal.
This work provides useful information about the potential use of
pectic polysaccharides for a product development for beverages taste
modulation, particularly related to astringency.
Acknowledgments
This work was supported by Fundação para a Ciência e Tecnologia
(FCT) by one fellowship (SFRH/BPD/112465/2015) and by the project
8
Carbohydrate Polymers 236 (2020) 116044
Fig. 6. Thermograms of titration of pectic
polysaccharides fractions CSP and WSP (6.8
and 5.8 mM galacturonic acid equivalents, re-
spectively) by PC fraction (35 and 30 mM ca-
techin equivalents, respectively): (top)
Thermogram of the titration of WSP/CSP by
PC; (bottom) Molar enthalpy change against a
PC/pectic polysaccharides ratio after peak in-
tegration concerning binding isotherm points
and fitting curve (line).
PTDC/AGR-TEC/6547/2014. The work was also supported by UID/
QUI/50006/2019 with funding from FCT/MEC through national funds
and by one Post-Doctoral fellowship funded by the project ref.
0377_IBERPHENOL_6_E from FEDER-Interreg España Portugal
Programme. Susana S. acknowledges the Post-Doctoral contract from
FCT.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116044.
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