Food Chemistry 159 (2014) 323–327

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evaluation of tannins interactions in grape (Vitis vinifera L.) skins

Laura Rustioni ⇑, Simone Fiori, Osvaldo Failla
Università degli Studi di Milano, CIRIVE – Centro Interdipartimentale di ricerca per l’innovazione in Viticoltura ed Enologia, via Celoria 2, I-20133 Milano, Italy

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

Article history: Tannins have a central role in grapevine berries both for their physiological and enological implications.
Received 20 January 2014 In the skin tissue they can be in vacuolar solution, or associated to the cell walls through weak or strong
Received in revised form 3 March 2014 physicochemical interactions. The present work aims to separate vacuolar, non-covalently and covalently
Accepted 8 March 2014
bonded tannins fractions. A specific extraction procedure was developed. A first extraction in ethanol at
Available online 17 March 2014
low temperature allowed the quantification of vacuolar tannins. An urea treatment followed by an
ethanol extraction at room temperature was able to separate non-covalently bonded compounds. Finally
Keywords:
an acid catalysis was used to break down proanthocyanidin covalent bonds. The method was validated on
Grapevine
Molecular interactions
ripe grape samples of three cultivars, on berries developed in two sun exposure conditions. The Ethephon
Spectrophotometric assays treatment effect was also evaluated. Beside the method development, a preliminary evaluation of the
Polyphenols cultivar, exposition and Ethephon treatment effects are discussed.
Proanthocyanidins Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction and the presence or absence of the 3-hydroxyl group (Dixon

Flavanols comprise monomers, oligomers and polymers, called
condensed tannins or proanthocyanidins. These compounds are
widespread throughout the plant kingdom, where they accumulate
in many different organs and tissues (Dixon, Xie, & Sharma, 2005).
In grapevines they are present in wood, stems, leaves, and fruits
(Terrier, Ollé, Verrès, & Cheynier, 2009). They are quantitatively
the most abundant secondary metabolites of grape berries, where
they are found in the hypodermal layers of the skin and the soft
parenchyma of the seed outer integument, between the epidermis
cuticle and the hard seed coat parenchyma (Adams, 2006). They
are partially extracted during winemaking, and they represent
one of the major qualitative factor in red wines because of their
implication in astringency, bitterness and color stability (Terrier
et al., 2009).
Since tannins are polymers of flavan-3-ol subunits, a very wide
range in molecular weight is possible (Kennedy, Saucier, & Glories,
2006; Xie & Dixon, 2005). They vary in size from dimers and
trimers up to oligomers with more than 30 subunits (Adams,
2006). Grape tannins are actually a very diverse set of biomole-
cules. Consequently, their structure depends upon the nature
(stereochemistry and hydroxylation pattern) of the flavan-3-ol
starter and extension units, the position and stereochemistry of
the linkage to the terminal unit, the degree of polymerization,
⇑ Corresponding author. Tel.: +39 0250316556; fax: +39 0250316553.
E-mail addresses: laura.rustioni@unimi.it (L. Rustioni), simone.fiori@unimi.it
(S. Fiori), osvaldo.failla@unimi.it (O. Failla).
et al., 2005; Xie & Dixon, 2005). In general, epicatechin is the most
abundant extension unit in grape tannins (Adams, 2006). Skin tan-
nins also contain epigallocatechin subunits whereas this flavan-3-
ol is missing in seed tannins. However, with respect at skin tannins
the smaller seed proanthocyanidins generally have a higher pro-
portion of epicatechin gallate in their subunits (Adams, 2006).
The structure and composition of grape proanthocyanidins have
been widely studied, however the tannin interactions in plant (and
particularly in grape) tissues is still not completely elucidated.
Tannins are amphipathic molecules, having both hydrophobic
aromatic rings and hydrophilic hydroxyl groups, allowing them
to bind simultaneously at several sites on the surface of other mol-
ecules (Hanlin, Hrmova, Harbertson, & Downey, 2010). In grape
skins, they could interact with proteins and cell wall polysaccha-
rides. The mechanism of tannin-protein binding involves hydrogen
bonding and hydrophobic interactions (Hanlin et al., 2010). Cell
wall polysaccharides also contain hydroxyl groups as well as glyco-
sidic oxygen atoms that have the ability to form hydrogen bonds
and hydrophobic interactions with tannin (Hanlin et al., 2010;
Renard, Baron, Guyot, & Drilleau, 2001). Moreover, polysaccharides
can interact with tannins also through covalent bonds (Kennedy,
Hayasaka, Vidal, Waters, & Jones, 2001).
Ripening processes (including partial cell wall degradation and
possible breakdown in compartmentalization) might be modulated
by specific plant growth regulators. Ethephon, sprayed in aqueous
solution, penetrates into the plant tissues, where it acts via libera-
tion of ethylene which is the active metabolite. Ethylene, in partic-
ular in climacteric fruit enhances the ripening processes. Grape is a

http://dx.doi.org/10.1016/j.foodchem.2014.03.027
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
324 L. Rustioni et al. / Food Chemistry 159 (2014) 323–327
‘‘non climacteric’’ fruit. However, ethylene effects, through a tran-
sient induction of several genes involved in grape ripening, phenols
and cell wall metabolism, have been fully demonstrated before
now (Böttcher & Davies, 2012).
In general, because tannins are located in the thick-walled
hypodermal cells of grape skin, in different cell compartments,
including vacuolar solution, plasma membrane and tonoplast, as
well as cell walls (Adams, 2006), the understanding of their inter-
actions with cell structural materials could have an important
impact for both the biological and the technological (extraction
during winemaking) implications.
The objectives of this work are: (i) to develop an analytical
method able to quantify the tannins interactions in grape skins,
and (ii) to preliminarily evaluate the impact of agronomical
variables (cultivar, bunch exposure and Ethephon treatment) on
the tannins repartition.
2. Material and methods
2.1. Chemicals and materials
Calibrations were performed by using standard compounds
(cyanidin chloride and catechin) from Sigma–Alderich without fur-
ther purifications. Solvents (ethanol, butanol, hydrochloric acid)
from Sigma–Alderich were used for extractions and reactions.
Urea, ferric ammonium sulfate and sodium molibdate used for
reactions were purchased from Sigma–Alderich.
Centrifugations were performed by a Megafuge 1.0 r (Heraeus).
A Jasco 7800 UV–Vis spectrophotometer was used to record
spectrophotometric data.
2.2. Plant material
Barbera (clone AT 84), Croatina (clone R2) and Nebbiolo (clone
R3 mic) grapes used in this experiment are grown in the same
germplasm collection vineyard, in the Regional Research Station
of Riccagioia (Lombardy region, northern Italy), already described
in Rustioni et al. (2013b).
The experiment was performed in 2010 on 30 grapevines for
each cultivar. With the purpose of obtaining two different expo-
sure growing conditions, the plants were divided in groups of 15
grapevines each, randomized in 5 plants block. In half of the plants,
at fruit-set, leaves were removed until the second distal node after
the last bunch of each shoot. Between veraison and ripening,
treatments were done spraying a 300 ppm Ethephon solution
weekly in half of the bunches for each exposure conditions.
2.3. Sampling
The extraction kinetics were evaluated on 8 samples. Skins of
three cultivars sampled at different stages of berry development
were used, with the purpose of maximizing the variability between
the samples:
 Nebbiolo, bunch closure stage (77 BBCH stage).
 Croatina, post-veraison (83 BBCH stage).
 Barbera, ripening (89 BBCH stage) (Meier, 2001).
Concerning the method development, Nebbiolo and Croatina
were analyzed in three biological replications, while Barbera was
only duplicated.
At ripening, three biological replications were collected from
each growing conditions of exposure and Ethephon treatment of
each cultivar. These samples were analyzed in two analytical
replications, by using the developed method. A total of 72 analysis
were performed.
Skins of fresh harvested grapes were directly separated and then
kept frozen (20 °C) until the extraction. Just before the analysis,
the skins were powdered in liquid nitrogen. Frozen samples were
directly suspended in the extraction solvent, to avoid the beginning
of oxidation reactions related to the defrosting process.
2.4. Extraction kinetics and analytical assays
All the analysis were performed on 1 g of skin powder samples.
To evaluate the extraction kinetics, consecutive extractions in
10 ml solvents were performed. Solvents were changed following
a specific timing. Concerning the vacuolar tannins, the extraction
was performed at low temperature (+4 °C) and ethanol was
changed 30, 120, 180, 240 min after the analysis beginning. It is
well known that tannins are highly soluble in alcoholic media. In
fact methanol and ethanol are capable of hydrogen-bonding and
dipole–dipole interactions with phenols (Murga, Ruiz, Beltràn, &
Cabezas, 2000). To quantify the hydrogen bonded tannins, a
treatment with 1 ml 6 M urea was performed for 45 min at room
temperature (Renard et al., 2001). After that, the extraction kinetics
were evaluated with ethanol at room temperature in continuous
shaking, changing solvent after 45, 75, 540, 1530 min. Finally,
covalent bonded tannins were quantify through an acid catalysis
at 100 °C following Porter, Hrstich, and Chan (1986). During all
the procedure, the suspension was centrifuged before each solvent
change, to better separate the obtained solution.
All the obtained 10 ml extracts were separately analyzed to
evaluate the extraction procedure. After that, the method was
simplified as shown in Table 1.
Phenolics were quantified applying the acid catalysis (Porter
et al., 1986), and the ortho-diphenol analysis (Duran Maestro, Borja
Padilla, Martin Martin, Fiestas Ros de Ursinos, & Alba Mendoza,
1991) methods.
Statistical analysis was performed by using SPSSÓ software
(PASW Statistics 18 version, SPSS Inc. Chicago, Illinois)
3. Results and discussion
3.1. Method development
3.1.1. Extraction kinetics
Aiming to the evaluation of the tannins interactions in the grape
skins, three consecutive extractions procedures were performed.
The objective was to separate vacuolar, non-covalently and cova-
lently bonded tannins. Fig. 1 shows the differential extraction
kinetics during the time.
The first extraction is made by pure ethanol at low temperature
(+4 °C). This solvent was selected because of the high solubility of
tannins in alcoholic media. In the other hand, the low temperature
should increase the strength of hydrogen bonds, limiting the
extraction of polyphenols interacting with the cell matrix.
On average, after 15 min the 22.14% of the total molecules were
extracted; after the successive 45 min an additional amount of the
13.18% was extracted. The following steps indicated that most of
the vacuolar phenolic compounds had been previously extracted,
in fact in the subsequent 150 min, only an extra quantity of
2.76% of compounds were extracted further, with the following
kinetic (+90 min:+1.81%; +30 min:+0.57%; +30 min:+0.38%).
Once the extraction lag-phase was obtained, a treatment with
6 M urea, a chaotropic reagent, was performed at room tempera-
ture, with the objective to disrupt week interactions, and especially
hydrogen bonds. Urea have been already used by Renard et al.
(2001) to characterize procyanidins-polysaccharides weak
 L. Rustioni et al. / Food Chemistry 159 (2014) 323–327 325

Table 1
Extraction procedure. The analysis was performed on 1 g of skin powder (obtained crashing the samples in liquid nitrogen) in two analytical replications.

Extraction Solvent ml Extraction condition Separation procedure Conservation until analysis

duration
1h Ethanol 15 +4 °C Centrifugate for 2 min at Separate the liquid part and mix the three extracts. Keep them at
2000 rpm 20 °C until analysis
1 h 30 min Ethanol 15 +4 °C Centrifugate for 2 min at
2000 rpm
30 min Ethanol 10 +4 °C Centrifugate for 2 min at
2000 rpm
1h Urea 6 M 1 Room temperature
2h Ethanol 10 Room temperature; Centrifugate for 2 min at Separate the liquid part and mix the three extracts. Keep them at
continuous shaking 2000 rpm 20 °C until analysis
18 h Ethanol 15 Room temperature; Centrifugate for 2 min at
continuous shaking 2000 rpm
3 h 30 min Ethanol 10 Room temperature; Centrifugate for 2 min at
continuous shaking 2000 rpm
1h Butanol:HCl 10 +100 °C Centrifugate for 2 min at Analyze the extract
95:5 2000 rpm

Stefanini, & Velasco, 2006), it cannot react with Mo, because the
ortho-diphenol moiety is missing.
The concentration, was than calculated using the formula
obtained by a calibration curve (Supplementary Information, SI1):

Catechin ðmg=lÞ ¼ ð216:31  E370nm þ 4:7966Þ  D

E370nm ¼ absorbance at 370 nm

Fig. 1. Differential extraction kinetics. Gray lines represent the kinetics obtained by
the eight samples analyzed (3 Nebbiolo, 3 Croatina, 2 Barbera). The black line shows
the average behavior.
D ¼ dilution factor
Proanthocyanidins (the common grape tannins), take their
name from the well-known property to produce pigments after
an acid catalysis (Dixon et al., 2005; Xie & Dixon, 2005). Thus, this
characteristic reaction was used to quantify the covalent bonded
molecules. A calibration curve was obtained using cyanidin
(Supplementary Information, SI2), obtaining the following formula:

Cyanidin chloride ðmg=lÞ ¼ ð16:956  E520nm þ 0:0989Þ  D

hydrogen bonds in apple. This addition allowed the extraction of a
high amount of compounds just after the treatment. In 25 min
57.27% of the total molecules were extracted. Keeping the extrac-
tion for other 965 min with ethanol at room temperature under
continuous shaking, only an extra amount of 2.35% of the total
compounds were extracted (+210 min:+1.57%; +405 min:+0.42%;
+525 min:+0.36%).
Finally, the acid catalysis provoked the break of the covalent
bonds among proanthocyanidins. In fact, as exploited by a classical
chemical assay for these compounds, as a result of acid hydrolysis,
the proanthocyanidins extension units are converted into colored
anthocyanidins (Dixon et al., 2005; Xie & Dixon, 2005). In half an
hour of extraction at 100 °C in acidic media, another 2.35% of com-
pounds were extracted.
Following these results, and aiming at a simplification of the
method, the extraction procedure was designed as shown in
Table 1.
3.1.2. Quantification
Our objective was to select high specific spectrophotometric
methods for tannins quantification.
The first two extracts were analyzed following the assay pro-
posed by Duran Maestro et al. (1991). It is based on the ortho-diphe-
nol property to interact with some cations (in this case Mo),
producing an evident change in the optical properties. This analysis
was selected, instead of the more common Folin-Chocalteau reac-
tion, because, beside tannins, anthocyanins are abundant phenolics
in grape skins. However, considering that malvidin-3-O-glucoside is
usually the most represented pigment (Mattivi, Guzzon, Vrhovsek,
E370nm ¼ absorbance at 520nm
D ¼ dilution factor
In this work, all the quantifications were performed by spectro-
photometric methods. However, in the future, more information
could be obtained by using HPLC assays such as the phloroglucin-
olysis (Kennedy & Jones, 2001). In this way new and interesting
information such as mDP or monomer proportion of the different
fractions could be obtained.
To compare properly the obtained data, results were expressed
in mol/g of skin, considering that each cyanidin chloride was it de-
rived from a flavan-3-ol subunit of the grape skin tannins. In this
way, the data obtained by different assays became comparable as
flavan-3-ol moles quantified in the three fractions. The repartition
of the tannins among the assumed cell compartment and physico-
chemical interactions was expressed as percentage of the total
content.
3.2. Ripened samples analysis
3.2.1. Method validation and expected variance components
The developed method was applied for studying tannins inter-
actions in ripe samples of Croatina, Nebbiolo and Barbera grapes,
with berries developed in two sunlight exposure conditions. The
Ethephon treatment effect on ripening bunches was also tested.
Table 2 shows the Expected Variance Components estimation,
referred, according to the experimental plan, to the effects of the
326 L. Rustioni et al. / Food Chemistry 159 (2014) 323–327

Table 2
Expected Variance Components (in percent on the total variance) among the tannins fractions (mol/g of skins). The analytical replication is responsible of a very low fraction of
the total variance.

Component Vacuolar tannins Non-covalent bonded tannins Covalent bonded tannins Tot
Cultivar 20.56 0.00 0.00 8.63
Bunch exposure 0.00 0.00 0.00 0.00
Ethephon treatment 5.84 12.97 37.85 12.82
Cultivar  Bunch exposure 15.97 22.66 21.01 21.27
Cultivar  Ethephon treatment 0.00 0.64 0.00 0.00
Bunch exposure  Ethephon treatment 0.00 8.29 0.00 0.00
Cultivar  Bunch exposure  Ethephon treatment 39.64 24.74 26.89 42.23
Biological replication 16.04 27.97 12.58 14.30
Analytical replication 1.95 2.74 1.67 0.75

main sources of variation and their interactions, on the total
variability among the analyzed samples.
It is possible to note that the analytical replications are respon-
sible of a very low variability, especially in the first extraction. As
expected, higher values were obtained concerning the biological
replications, underlining the importance of the sampling proce-
dure in the field.
The variability between the cultivars affected mainly the vacu-
olar tannins. On the other hands, the Ethephon treatment during
ripening had a high impact on the covalent bonded compounds.
The bunch exposure condition had an impact on the data variabil-
ity (15–23%) only when considered in interaction with the culti-
var. An important role was also taken by the interactions between
the three considered sources of variations, underlining the differ-
ent cultivar reactivity in respect to the tested ripening conditions.
3.2.2. Cultivar, exposition and ethephon treatment effects
Table 3 shows the tannins content and percentage repartition
with respect to the cultivar, the bunch exposure, and the Ethephon
treatment. In general more than half of the total tannins were lo-
cated in the vacuolar solution. About one third of the molecules
were interacting through week interactions. Only a low proportion
(7–18%) of proanthocyadins were linked to the matrix through
covalent bonds. However, an underestimation of the acid catalysis
assay need to be taken into consideration. In fact, only the extension
subunits are converted in anthocyanidins, thus the flavan-3-ols ter-
minal subunits are not considered. However, as the average size of
skin tannins is known to be quite large (i.e., much larger than seed
tannins) (Adams, 2006), this index should be enough consistent.
Table 3a shows the differences between the three cultivars ana-
lyzed. In general, higher tannins contents were found in Croatina,

Table 3
Tannins content and percentage repartition with respect to the cultivar (a), the bunch exposure (b), and the Ethephon treatment (c). In each table and for each column the mean
values followed by the same letter are not statistically different (P = 5%).

Non-covalent bonded Covalent bonded

a Vacuolar tannins
tannins tannins
mol/g of skin mol/g of skin mol/g of skin
cultivar % % %
14.2 b 8.6 c 3.2 b
Barbera 54 b 33 a 12 b
44.0 a 19.6 a 4.7 a
Croatina 64 a 29 a 7c
17.3 b 10.9 b 5.5 a
Nebbiolo 50 b 32 a 18 a

Non-covalent bonded Covalent bonded

b Vacuolar tannins
tannins tannins
mol/g of skin mol/g of skin mol/g of skin
exposure % % %
22.71 b 12.53 a 4.31 b
shaded 56 a 32 a 12 a
27.72 a 13.65 a 4.72 a
exposed 57 a 31 a 13 a

Non-covalent bonded Covalent bonded

c Vacuolar tannins
tannins tannins
mol/g of skin mol/g of skin mol/g of skin
treatment % % %
24.95 a 11.68 a 3.35 b
Ethephon 59 a 31 a 9b
25.47 a 14.49 a 5.68 a
No treatment 53 b 32 a 14 a
 L. Rustioni et al. / Food Chemistry 159 (2014) 323–327
followed by Nebbiolo. The lowest tannins concentration was found
in Barbera skins. When considering the repartition of the mole-
cules among the interaction types, it is possible to note that a sim-
ilar proportion of tannins were interacting through non-covalent
bonds (about 30%), while significant differences were found be-
tween vacuolar tannins and covalent bonded proanthocyanidins
among the three cultivars. This information could be of interest
for the winemakers, to support the maceration management. A
higher proportion of vacuolar tannins (such as in the case of
Croatina grapes), could facilitate the extraction during the first
winemaking steps. It is interesting to note that, in their major
production areas, traditionally Croatina is vinified using short mac-
eration times, while Nebbiolo wines are usually produced by long
maceration techniques.
The canopy management and the bunch exposure during berry
development are already known to affect the phenolic accumula-
tion. As an example, our research group already studied the radia-
tion effect on anthocyanin composition (Rustioni, Rossoni, Cola,
Mariani, & Failla, 2011b; Rustioni, Rossoni, Failla, & Scienza,
2013a) and extractability (Rustioni, Rossoni, Calatroni, & Failla,
2011a). However, in the present work, no significant differences
were found in the tannins interactions repartition. Nevertheless,
exposed grapes had a small but significant higher concentration
of vacuolar and covalent bonded tannins (Table 3b).
Table 3c shows the treatment effect on the tannins content and
repartition. It is interesting to note that the Ethephon treatment
modify the proportion between vacuolar and covalent bonded
tannins, mainly due to the decrease of bonded molecules as a
consequence of the treatment. This could be due to the Ethephon
effect on the cell walls metabolism which increased the degree of
polysaccharides degradation and consequently the tannins release.
4. Conclusion
Tannins have a crucial role both for the productive and the phys-
iological point of view. Beside their concentration, information con-
cerning tannins interactions in grape skin tissue could support the
oenological industry concerning the wine-making management
techniques. In the present work, a method was developed to sepa-
rate and quantify vacuolar, hydrogen and covalent bonded mole-
cules. The method was validated by using three cultivars grape
samples developed in different conditions (sun exposition and Eth-
ephon treatment). A first evaluation of the varietal and management
effects on the tannins interactions was also discussed. The results
encourage the application of this protocol in further studies con-
cerning plant physiology and agricultural/food production.
Acknowledgements
We thanks Dr. Mara Rossoni for technical support.
This research paper is published in the framework of the project
PRIN 2008 supported by the Italian Ministry of Education, University
and Research.
327
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
03.027.
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