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Article
Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma Loss Simone Vincenzi, Annarita Panighel, Diana Gazzola, Riccardo Flamini, and Andrea Curioni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505657h • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 15, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26
Journal of Agricultural and Food Chemistry
1
Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma
2
Loss
3 4
Simone Vincenzi*,1, Annarita Panighel2, Diana Gazzola1, Riccardo Flamini2, and Andrea Curioni1
5
6
1. Department of Agronomy, Food, Natural Resources, Animals and the Environment (DAFNAE),
7
University of Padova, Viale XXVIII Aprile, 14, 31015 Conegliano (TV), Italy
8
2. Consiglio per la Ricerca e la Sperimentazione in Agricoltura–Centro di Ricerca per la Viticoltura
9
(CRA-VIT) Viale XXVIII Aprile 26, 31015 Conegliano (TV), Italy
10 11 12
*e-mail: [email protected]; Fax: +39 0438 453744; Phone : +39 0438 453052
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
13
ABSTRACT
14
The wine aroma loss as a consequence of treatments with bentonite is due to the occurrence of
15
multiple interaction mechanisms. In addition to a direct effect of bentonite, the removal of aroma
16
compounds bound to protein components adsorbed by the clay has been hypothesized, but never
17
demonstrated. We studied the effect of bentonite addition on total wine aroma compounds
18
(extracted from Muscat wine) in a model solution in the absence and presence of total and purified
19
(Thaumatin-like proteins and chitinase) wine proteins. The results showed that in general bentonite
20
alone has a low effect on the loss of terpenes, but removed ethyl esters and fatty acids. The presence
21
of wine proteins in the solution treated with bentonite tended to increase the loss of esters with the
22
longest carbon chains (from ethyl octanoate to ethyl decanoate), and this was significant when the
23
purified proteins were used. The results here reported suggest that hydrophobicity can be one of the
24
driving forces involved in the interaction of aromas with both bentonite and proteins.
25 26
Keywords: wine aroma, bentonite, wine proteins, ethyl esters, terpenes
27
2 ACS Paragon Plus Environment
Page 2 of 26
Page 3 of 26
Journal of Agricultural and Food Chemistry
28
INTRODUCTION
29
The presence of haze in bottled white wines results in a serious quality defect because turbidity
30
makes the wine undesirable for consumers. Wine proteins, which have the tendency to insolubilize
31
during wine storage1-3, are the main cause for this defect. The majority of wine proteins derive from
32
grapes, and, in particular, the haze-forming proteins have been found by several authors to be grape
33
specific4-6. These proteins have been identified as grape pathogenesis related and, due to their
34
resistance to proteolysis and their stability at acidic pH3, they are able to persist throughout the
35
winemaking process.
36
The addition of bentonite, a montmorillonite clay, is universally employed in the wine industry for
37
the prevention of white wine protein hazing. Bentonite, which carries a net negative charge, interact
38
electrostatically with the positively charged wine proteins, which results in their removal from the
39
wine. However, bentonite action has been shown to be non specific for proteins, as it also removes
40
other charged species or aggregates7 and also a direct interaction between bentonite and aroma
41
compounds has been demonstrated in model solutions containing selected aromatic molecules8.
42
This would contribute to the loss of sensorial quality that is often claimed for bentonite treated
43
white wines.
44
However in addition to the direct bentonite-aroma compounds interaction mechanism, the
45
possibility that part of the aroma compound are removed because they are bound to the wine
46
proteins cannot be excluded.
47
Many other papers attempted to investigate bentonite-aroma interaction, but most of them were
48
performed by headspace analysis or in model solution containing only some selected aroma
49
molecules representative of different chemical classes9-11.
50
In addition, the synergistic effect of proteins and bentonite in removing aroma compounds has been
51
supposed by many authors10-12 but this phenomenon has been demonstrated only once13. In that case
52
the effect on selected aromas (β-ionone and γ-decalactone) was, however, studied in a model
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
53
solution in the absence of ethanol, with ovalbumin as standard protein and with a protein
54
concentration ten times higher than that normally present in wine.
55
A more detailed study of the interactions among aroma compounds, bentonite and wine proteins is
56
then needed to understand the nature of mechanisms involved and to suggest the best practices for
57
bentonite treatment in order to preserve as much as possible wine aroma. In order to assess the
58
influence of wine proteins on the aroma composition of wines treated with bentonite, in this paper
59
the effect of bentonite fining in a wine model solution containing the aroma compounds extracted
60
from wine was studied in the absence and presence of total and purified wine proteins.
4 ACS Paragon Plus Environment
Page 4 of 26
Page 5 of 26
61
Journal of Agricultural and Food Chemistry
MATERIALS AND METHODS
62 63
Chemicals. Standards of 1-heptanol and 1-decanol were purchased from Carlo Erba Reagents
64
(Milan, Italy). Ethyl decanoate and ethyl dodecanoate were purchased from Fluka (Sigma-Aldrich
65
srl, Milan, Italy), ethyl hexanoate from B.H.D Laboratory Chemical Division (Poole, England),
66
ethyl octanoate from Eastman Organic Chemicals (Rochester, US).
67
Extraction of proteins and polysaccharides. For the extraction of wine macromolecules an
68
untreated Manzoni Bianco wine (vintage 2011) produced in the Veneto region was used. This type
69
of wine was chosen because it contains a high protein amount14. Macromolecules were purified
70
following the preparative method proposed by Van Sluyter et al.15 with some modifications. Ten
71
liters of wine were treated with 4 g/L PVPP (Sigma-Aldrich, Milan, Italy) and 1.5 g/L charcoal
72
(Sigma-Aldrich, Milan, Italy) for 24 h at 15 °C in order to remove most of the polyphenols. The
73
wine was then filtered with GF/A filters (Whatman, Kent, UK) and finally at 0.45 µm with cellulose
74
acetate membranes (Sartorius AG, Göttingen, Germany) and brought at pH 3.0 with HCl. Aliquots
75
of 5 liters were loaded at 8 mL/min on a S-Sepharose column (5 x 14 cm) (Pharmacia, Uppsala,
76
Sweden) previously equilibrated in trisodium citrate buffer 30 mM pH 3.0. After washing with two
77
column volumes with the same buffer, proteins were eluted with a gradient of NaCl from 0 to 1 M.
78
Both the flow through (containing the unbound material, i. e. mainly polysaccharides) and the pool
79
of the eluted peaks (representing the total proteins) were ultrafiltered at 3 kDa with an Amicon 8400
80
apparatus (Sartorius), then dialyzed on regenerate cellulose membrane (3.5 kDa, Cellu-Sep®)
81
against water and finally freeze dried. Five litres of the same wine were used for the purification of
82
two main wine proteins (Chitinase and the Thaumatin-like protein VVTL1). In this case the
83
fractions obtained after cation exchange separation on S-Sepharose were diluted in 1.25 M
84
ammonium sulphate and further purified by Hydrophobic Interaction Chromatography (HIC) on a
85
Phenyl-Sepharose HP column (GE-Healthcare) as follows. After washing the column with 1.25 M
86
ammonium sulfate in 50 mM sodium citrate, pH 5.0, proteins were eluted with a 110 min linear 5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
87
gradient up to 100% 50 mM sodium citrate, pH 5.0. The peaks obtained with this second step were
88
dialyzed against water (3.5 kDa MWCO), and freeze dried. The purity and identity of the fractions
89
were assessed by RP-HPLC15 and SDS-PAGE16.
90
Aroma extraction. Fermentative compounds and grape volatiles were extracted from a dry
91
Moscato bianco wine (vintage 2011) produced in the Colli Euganei (Veneto, Italy) not previously
92
treated with bentonite by using the method previously reported for wines and model wine
93
solutions17. This type of wine was chosen because of its high content of primary aroma compounds.
94
Aliquots of 500 mL were spiked with 4 mL of 1-heptanol (442 mg/L) as the internal standard, and
95
extracted 3×50 mL with dichloromethane (Carlo Erba, Milano. Italy). Extraction was carried out
96
under agitation for 15 minutes each time. The organic phase containing the aroma compounds was
97
collected and washed 3×30 mL with 5% NaHCO3 to remove the acidic compounds by salification
98
and shift into aqueous phase. After dehydration with anhydrous sodium sulfate, dichloromethane
99
was filtered on blue ribbon filters 589/3 (Whatman, UK). The extract was concentrated first using a
100
Vigreux column and finally under nitrogen flow. This extract was then used for the reconstitution
101
experiments in model wine solution.
102
Fifty mL of the clear supernatant of each reconstituted sample were used for the extraction of the
103
aroma compounds remaining after bentonite treatment. The samples were spiked with 400 µL of 1-
104
decanol 450 mg/L as the internal standard and extracted 3×15 mL with dichloromethane. After
105
dehydration with anhydrous sodium sulfate and filtration as previously described, the extracts were
106
concentrated with a Vigreux column (l=40 cm) to approximately 3 mL and adjusted to 1 mL under
107
nitrogen flow before GC/MS analysis.
108
Stability test. The purified proteins were suspended at 100 mg/L in model wine (tartaric acid 5 g/L,
109
ethanol 12%, pH 3.2) or in a ultrafiltered Manzoni bianco wine (3.5 kDa). Bentonite Nucleobent
110
(EVER, Pramaggiore, Italy) was prepared at 5% (w/v) in water for 24 h, and then different amounts
111
(5, 10, 15 g/hL, final concentrations) were added to the wine protein solution. After 1 h at room
112
temperature the solutions were filtered at 0.45 µm on cellulose acetate membrane (Sartorius). In 6 ACS Paragon Plus Environment
Page 6 of 26
Page 7 of 26
Journal of Agricultural and Food Chemistry
113
order to assess the bentonite dose necessary to reach the protein stability, the protein solutions
114
prepared in ultrafiltered wine were subjected to the heat test as reported by Pocock and Rankine18.
115
Briefly, the samples were heated at 80°C for 6 h, followed by incubation at 4°C for 12 h, then the
116
turbidity was measured with a nefelometer (HACH). When the difference in the turbidity measured
117
before and after the heating was less than 2 NTU the sample could be considered stabile. For the
118
samples prepared in model solution the dose of bentonite necessary for stabilization was instead
119
calculated as the amount able to reduce the protein content below 20 mg/L.
120
Determination of the protein content. The protein content was measured according to Vincenzi et
121
al.19. Firstly, proteins were precipitated from 1 mL of wine with 10 µL of SDS 10% followed by
122
250 µL of KCl 1M. The pellets were dissolved in 1 mL of distilled water and quantified by the
123
Smith method20, using the BCA-200 protein assay kit (Pierce, Rockford, IL) according to the
124
manufacturer instructions. The calibration curve was prepared by using serial dilution of bovine
125
serum albumin (BSA, Sigma, Milan, Italy) in water.
126
Electrophoresis. Electrophoretic analyses were performed according to Laemmli21. Samples to be
127
analyzed were dissolved in a 0.5 M Tris-HCl pH 6.8 buffer containing 15% (v/v) glycerol (Sigma-
128
Aldrich, Milano, Italy), 1.5 % (w/v) SDS (Bio-Rad laboratories, Segrate, Italy) and 4% β-
129
mercaptoethanol (Sigma-Aldrich, Milano, Italy) and heated at 100°C for 5 minutes before loading.
130
For both fractions (bound and unbound material) 20 µg of lyophilized powder were loaded in each
131
lane. Electrophoresis was performed in a Mini-Protean III apparatus (Bio-Rad, Hercules, CA) with
132
T = 14% (acrylamide/N, N’ metylen-bisacrylamide 29:1; Fluka, Milan, Italy) gels. The molecular
133
weight standard proteins were Broad Range Molecular Weight Markers (Bio-Rad, Hercules, CA).
134
After electrophoresis, gels were stained for 18 h with colloidal Coomassie and then destained with
135
water for 24 h. The Periodic Acid-Schiff (PAS) method was used to stain glycoproteins as described
136
by Segrest and Jackson22.
137
Reconstitution experiments. The concentrated extract was diluted in model wine to reach a
138
concentration of the aroma compounds equal to that found in the original Moscato wine. Ten g/hL 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
139
of bentonite alone or 100 mg/L of proteins followed by 10 g/hL of bentonite were added to 55 mL
140
of this solution. An additional experiment with 100 mg/L of proteins and 200 mg/L of the S-
141
Sepharose unbound fraction followed by 10 g/hL bentonite was performed. Each experiment was
142
done in triplicate.
143
The reconstituted samples were stored at 10 °C (in closed containers) for 12 h and centrifuged
144
(5000g, 5 minutes, 15 °C), then 50 mL of clear supernatant were recovered and used for the aroma
145
extraction. A second experiment was performed using four selected ethyl esters instead of the total
146
aroma extract. Esters were ethyl hexanoate, octanoate, decanoate and dodecanoate at 1167, 1102,
147
1035 and 1145 µg/L, respectively dissolved in model wine. In this case the treatments were
148
performed as above, but adding the purified wine chitinase and Thaumatin-Like Protein (TLP)
149
separately (both at 100 mg/L) before treating the solution with 10 g/hL of bentonite. Each treatment
150
was done in triplicate.
151
GC/MS analysis. Gas chromatography/mass spectrometry (GC/MS) analysis was performed using
152
a 6850 gas chromatography system (Agilent Technologies, Santa Clara, CA, US), equipped with a
153
fused silica HP-INNOWax polyethylene glycol capillary column (30 m × 0.25 mm, 0.25 µm i.d.)
154
(Agilent Technologies, Santa Clara, CA, US), coupled with HP 5975C mass spectrometer and
155
7693A automatic liquid sampler injector (Agilent Technologies, Santa Clara, CA, US). Oven
156
temperature program: 40 °C isothermal for 1 min, heating 2 °C/min until 160 °C, 3 °C/min until
157
230 °C, 230 °C isothermal for 15 min. Other experimental conditions: injector temperature, 230 °C;
158
carrier gas, helium at flow rate of 1.2 mL/min; sample volume injected, 1 µL splitless injection;
159
transfer line temperature, 250 °C; quadrupole temperature, 150 °C; mass range, m/z 35-550.
160
Compounds identification was performed by using the spectral libraries NIST Mass (rev08)
161
Spectral Database and the homemade CRA-VIT database ESTRATTI. For some compounds, the
162
identification was confirmed on the basis of GC retention times of pure standards. For the other
163
compounds the Kovats retention index on PEG stationary phase was calculated by using a standard
8 ACS Paragon Plus Environment
Page 8 of 26
Page 9 of 26
Journal of Agricultural and Food Chemistry
164
mixture of C11-C32 aliphatic hydrocarbons. The concentration of compounds was calculated as
165
µg/L of internal standard 1-decanol.
166
Statistical Analysis. Data were analyzed by XLSTAT program. Significance was determined at
167
P< 0.05 or P
Article
Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma Loss Simone Vincenzi, Annarita Panighel, Diana Gazzola, Riccardo Flamini, and Andrea Curioni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505657h • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 15, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26
Journal of Agricultural and Food Chemistry
1
Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma
2
Loss
3 4
Simone Vincenzi*,1, Annarita Panighel2, Diana Gazzola1, Riccardo Flamini2, and Andrea Curioni1
5
6
1. Department of Agronomy, Food, Natural Resources, Animals and the Environment (DAFNAE),
7
University of Padova, Viale XXVIII Aprile, 14, 31015 Conegliano (TV), Italy
8
2. Consiglio per la Ricerca e la Sperimentazione in Agricoltura–Centro di Ricerca per la Viticoltura
9
(CRA-VIT) Viale XXVIII Aprile 26, 31015 Conegliano (TV), Italy
10 11 12
*e-mail: [email protected]; Fax: +39 0438 453744; Phone : +39 0438 453052
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
13
ABSTRACT
14
The wine aroma loss as a consequence of treatments with bentonite is due to the occurrence of
15
multiple interaction mechanisms. In addition to a direct effect of bentonite, the removal of aroma
16
compounds bound to protein components adsorbed by the clay has been hypothesized, but never
17
demonstrated. We studied the effect of bentonite addition on total wine aroma compounds
18
(extracted from Muscat wine) in a model solution in the absence and presence of total and purified
19
(Thaumatin-like proteins and chitinase) wine proteins. The results showed that in general bentonite
20
alone has a low effect on the loss of terpenes, but removed ethyl esters and fatty acids. The presence
21
of wine proteins in the solution treated with bentonite tended to increase the loss of esters with the
22
longest carbon chains (from ethyl octanoate to ethyl decanoate), and this was significant when the
23
purified proteins were used. The results here reported suggest that hydrophobicity can be one of the
24
driving forces involved in the interaction of aromas with both bentonite and proteins.
25 26
Keywords: wine aroma, bentonite, wine proteins, ethyl esters, terpenes
27
2 ACS Paragon Plus Environment
Page 2 of 26
Page 3 of 26
Journal of Agricultural and Food Chemistry
28
INTRODUCTION
29
The presence of haze in bottled white wines results in a serious quality defect because turbidity
30
makes the wine undesirable for consumers. Wine proteins, which have the tendency to insolubilize
31
during wine storage1-3, are the main cause for this defect. The majority of wine proteins derive from
32
grapes, and, in particular, the haze-forming proteins have been found by several authors to be grape
33
specific4-6. These proteins have been identified as grape pathogenesis related and, due to their
34
resistance to proteolysis and their stability at acidic pH3, they are able to persist throughout the
35
winemaking process.
36
The addition of bentonite, a montmorillonite clay, is universally employed in the wine industry for
37
the prevention of white wine protein hazing. Bentonite, which carries a net negative charge, interact
38
electrostatically with the positively charged wine proteins, which results in their removal from the
39
wine. However, bentonite action has been shown to be non specific for proteins, as it also removes
40
other charged species or aggregates7 and also a direct interaction between bentonite and aroma
41
compounds has been demonstrated in model solutions containing selected aromatic molecules8.
42
This would contribute to the loss of sensorial quality that is often claimed for bentonite treated
43
white wines.
44
However in addition to the direct bentonite-aroma compounds interaction mechanism, the
45
possibility that part of the aroma compound are removed because they are bound to the wine
46
proteins cannot be excluded.
47
Many other papers attempted to investigate bentonite-aroma interaction, but most of them were
48
performed by headspace analysis or in model solution containing only some selected aroma
49
molecules representative of different chemical classes9-11.
50
In addition, the synergistic effect of proteins and bentonite in removing aroma compounds has been
51
supposed by many authors10-12 but this phenomenon has been demonstrated only once13. In that case
52
the effect on selected aromas (β-ionone and γ-decalactone) was, however, studied in a model
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
53
solution in the absence of ethanol, with ovalbumin as standard protein and with a protein
54
concentration ten times higher than that normally present in wine.
55
A more detailed study of the interactions among aroma compounds, bentonite and wine proteins is
56
then needed to understand the nature of mechanisms involved and to suggest the best practices for
57
bentonite treatment in order to preserve as much as possible wine aroma. In order to assess the
58
influence of wine proteins on the aroma composition of wines treated with bentonite, in this paper
59
the effect of bentonite fining in a wine model solution containing the aroma compounds extracted
60
from wine was studied in the absence and presence of total and purified wine proteins.
4 ACS Paragon Plus Environment
Page 4 of 26
Page 5 of 26
61
Journal of Agricultural and Food Chemistry
MATERIALS AND METHODS
62 63
Chemicals. Standards of 1-heptanol and 1-decanol were purchased from Carlo Erba Reagents
64
(Milan, Italy). Ethyl decanoate and ethyl dodecanoate were purchased from Fluka (Sigma-Aldrich
65
srl, Milan, Italy), ethyl hexanoate from B.H.D Laboratory Chemical Division (Poole, England),
66
ethyl octanoate from Eastman Organic Chemicals (Rochester, US).
67
Extraction of proteins and polysaccharides. For the extraction of wine macromolecules an
68
untreated Manzoni Bianco wine (vintage 2011) produced in the Veneto region was used. This type
69
of wine was chosen because it contains a high protein amount14. Macromolecules were purified
70
following the preparative method proposed by Van Sluyter et al.15 with some modifications. Ten
71
liters of wine were treated with 4 g/L PVPP (Sigma-Aldrich, Milan, Italy) and 1.5 g/L charcoal
72
(Sigma-Aldrich, Milan, Italy) for 24 h at 15 °C in order to remove most of the polyphenols. The
73
wine was then filtered with GF/A filters (Whatman, Kent, UK) and finally at 0.45 µm with cellulose
74
acetate membranes (Sartorius AG, Göttingen, Germany) and brought at pH 3.0 with HCl. Aliquots
75
of 5 liters were loaded at 8 mL/min on a S-Sepharose column (5 x 14 cm) (Pharmacia, Uppsala,
76
Sweden) previously equilibrated in trisodium citrate buffer 30 mM pH 3.0. After washing with two
77
column volumes with the same buffer, proteins were eluted with a gradient of NaCl from 0 to 1 M.
78
Both the flow through (containing the unbound material, i. e. mainly polysaccharides) and the pool
79
of the eluted peaks (representing the total proteins) were ultrafiltered at 3 kDa with an Amicon 8400
80
apparatus (Sartorius), then dialyzed on regenerate cellulose membrane (3.5 kDa, Cellu-Sep®)
81
against water and finally freeze dried. Five litres of the same wine were used for the purification of
82
two main wine proteins (Chitinase and the Thaumatin-like protein VVTL1). In this case the
83
fractions obtained after cation exchange separation on S-Sepharose were diluted in 1.25 M
84
ammonium sulphate and further purified by Hydrophobic Interaction Chromatography (HIC) on a
85
Phenyl-Sepharose HP column (GE-Healthcare) as follows. After washing the column with 1.25 M
86
ammonium sulfate in 50 mM sodium citrate, pH 5.0, proteins were eluted with a 110 min linear 5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
87
gradient up to 100% 50 mM sodium citrate, pH 5.0. The peaks obtained with this second step were
88
dialyzed against water (3.5 kDa MWCO), and freeze dried. The purity and identity of the fractions
89
were assessed by RP-HPLC15 and SDS-PAGE16.
90
Aroma extraction. Fermentative compounds and grape volatiles were extracted from a dry
91
Moscato bianco wine (vintage 2011) produced in the Colli Euganei (Veneto, Italy) not previously
92
treated with bentonite by using the method previously reported for wines and model wine
93
solutions17. This type of wine was chosen because of its high content of primary aroma compounds.
94
Aliquots of 500 mL were spiked with 4 mL of 1-heptanol (442 mg/L) as the internal standard, and
95
extracted 3×50 mL with dichloromethane (Carlo Erba, Milano. Italy). Extraction was carried out
96
under agitation for 15 minutes each time. The organic phase containing the aroma compounds was
97
collected and washed 3×30 mL with 5% NaHCO3 to remove the acidic compounds by salification
98
and shift into aqueous phase. After dehydration with anhydrous sodium sulfate, dichloromethane
99
was filtered on blue ribbon filters 589/3 (Whatman, UK). The extract was concentrated first using a
100
Vigreux column and finally under nitrogen flow. This extract was then used for the reconstitution
101
experiments in model wine solution.
102
Fifty mL of the clear supernatant of each reconstituted sample were used for the extraction of the
103
aroma compounds remaining after bentonite treatment. The samples were spiked with 400 µL of 1-
104
decanol 450 mg/L as the internal standard and extracted 3×15 mL with dichloromethane. After
105
dehydration with anhydrous sodium sulfate and filtration as previously described, the extracts were
106
concentrated with a Vigreux column (l=40 cm) to approximately 3 mL and adjusted to 1 mL under
107
nitrogen flow before GC/MS analysis.
108
Stability test. The purified proteins were suspended at 100 mg/L in model wine (tartaric acid 5 g/L,
109
ethanol 12%, pH 3.2) or in a ultrafiltered Manzoni bianco wine (3.5 kDa). Bentonite Nucleobent
110
(EVER, Pramaggiore, Italy) was prepared at 5% (w/v) in water for 24 h, and then different amounts
111
(5, 10, 15 g/hL, final concentrations) were added to the wine protein solution. After 1 h at room
112
temperature the solutions were filtered at 0.45 µm on cellulose acetate membrane (Sartorius). In 6 ACS Paragon Plus Environment
Page 6 of 26
Page 7 of 26
Journal of Agricultural and Food Chemistry
113
order to assess the bentonite dose necessary to reach the protein stability, the protein solutions
114
prepared in ultrafiltered wine were subjected to the heat test as reported by Pocock and Rankine18.
115
Briefly, the samples were heated at 80°C for 6 h, followed by incubation at 4°C for 12 h, then the
116
turbidity was measured with a nefelometer (HACH). When the difference in the turbidity measured
117
before and after the heating was less than 2 NTU the sample could be considered stabile. For the
118
samples prepared in model solution the dose of bentonite necessary for stabilization was instead
119
calculated as the amount able to reduce the protein content below 20 mg/L.
120
Determination of the protein content. The protein content was measured according to Vincenzi et
121
al.19. Firstly, proteins were precipitated from 1 mL of wine with 10 µL of SDS 10% followed by
122
250 µL of KCl 1M. The pellets were dissolved in 1 mL of distilled water and quantified by the
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Smith method20, using the BCA-200 protein assay kit (Pierce, Rockford, IL) according to the
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manufacturer instructions. The calibration curve was prepared by using serial dilution of bovine
125
serum albumin (BSA, Sigma, Milan, Italy) in water.
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Electrophoresis. Electrophoretic analyses were performed according to Laemmli21. Samples to be
127
analyzed were dissolved in a 0.5 M Tris-HCl pH 6.8 buffer containing 15% (v/v) glycerol (Sigma-
128
Aldrich, Milano, Italy), 1.5 % (w/v) SDS (Bio-Rad laboratories, Segrate, Italy) and 4% β-
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mercaptoethanol (Sigma-Aldrich, Milano, Italy) and heated at 100°C for 5 minutes before loading.
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For both fractions (bound and unbound material) 20 µg of lyophilized powder were loaded in each
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lane. Electrophoresis was performed in a Mini-Protean III apparatus (Bio-Rad, Hercules, CA) with
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T = 14% (acrylamide/N, N’ metylen-bisacrylamide 29:1; Fluka, Milan, Italy) gels. The molecular
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weight standard proteins were Broad Range Molecular Weight Markers (Bio-Rad, Hercules, CA).
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After electrophoresis, gels were stained for 18 h with colloidal Coomassie and then destained with
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water for 24 h. The Periodic Acid-Schiff (PAS) method was used to stain glycoproteins as described
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by Segrest and Jackson22.
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Reconstitution experiments. The concentrated extract was diluted in model wine to reach a
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concentration of the aroma compounds equal to that found in the original Moscato wine. Ten g/hL 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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of bentonite alone or 100 mg/L of proteins followed by 10 g/hL of bentonite were added to 55 mL
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of this solution. An additional experiment with 100 mg/L of proteins and 200 mg/L of the S-
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Sepharose unbound fraction followed by 10 g/hL bentonite was performed. Each experiment was
142
done in triplicate.
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The reconstituted samples were stored at 10 °C (in closed containers) for 12 h and centrifuged
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(5000g, 5 minutes, 15 °C), then 50 mL of clear supernatant were recovered and used for the aroma
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extraction. A second experiment was performed using four selected ethyl esters instead of the total
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aroma extract. Esters were ethyl hexanoate, octanoate, decanoate and dodecanoate at 1167, 1102,
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1035 and 1145 µg/L, respectively dissolved in model wine. In this case the treatments were
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performed as above, but adding the purified wine chitinase and Thaumatin-Like Protein (TLP)
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separately (both at 100 mg/L) before treating the solution with 10 g/hL of bentonite. Each treatment
150
was done in triplicate.
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GC/MS analysis. Gas chromatography/mass spectrometry (GC/MS) analysis was performed using
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a 6850 gas chromatography system (Agilent Technologies, Santa Clara, CA, US), equipped with a
153
fused silica HP-INNOWax polyethylene glycol capillary column (30 m × 0.25 mm, 0.25 µm i.d.)
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(Agilent Technologies, Santa Clara, CA, US), coupled with HP 5975C mass spectrometer and
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7693A automatic liquid sampler injector (Agilent Technologies, Santa Clara, CA, US). Oven
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temperature program: 40 °C isothermal for 1 min, heating 2 °C/min until 160 °C, 3 °C/min until
157
230 °C, 230 °C isothermal for 15 min. Other experimental conditions: injector temperature, 230 °C;
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carrier gas, helium at flow rate of 1.2 mL/min; sample volume injected, 1 µL splitless injection;
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transfer line temperature, 250 °C; quadrupole temperature, 150 °C; mass range, m/z 35-550.
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Compounds identification was performed by using the spectral libraries NIST Mass (rev08)
161
Spectral Database and the homemade CRA-VIT database ESTRATTI. For some compounds, the
162
identification was confirmed on the basis of GC retention times of pure standards. For the other
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compounds the Kovats retention index on PEG stationary phase was calculated by using a standard
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Journal of Agricultural and Food Chemistry
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mixture of C11-C32 aliphatic hydrocarbons. The concentration of compounds was calculated as
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µg/L of internal standard 1-decanol.
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Statistical Analysis. Data were analyzed by XLSTAT program. Significance was determined at
167
P< 0.05 or P
