Study of Combined Effect of Proteins and Bentonite Fining on the Wine

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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

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Journal of Agricultural and Food Chemistry

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Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma

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Loss

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Simone Vincenzi*,1, Annarita Panighel2, Diana Gazzola1, Riccardo Flamini2, and Andrea Curioni1

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1. Department of Agronomy, Food, Natural Resources, Animals and the Environment (DAFNAE),

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University of Padova, Viale XXVIII Aprile, 14, 31015 Conegliano (TV), Italy

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2. Consiglio per la Ricerca e la Sperimentazione in Agricoltura–Centro di Ricerca per la Viticoltura

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(CRA-VIT) Viale XXVIII Aprile 26, 31015 Conegliano (TV), Italy

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*e-mail: [email protected]; Fax: +39 0438 453744; Phone : +39 0438 453052

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ABSTRACT

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The wine aroma loss as a consequence of treatments with bentonite is due to the occurrence of

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multiple interaction mechanisms. In addition to a direct effect of bentonite, the removal of aroma

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compounds bound to protein components adsorbed by the clay has been hypothesized, but never

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demonstrated. We studied the effect of bentonite addition on total wine aroma compounds

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(extracted from Muscat wine) in a model solution in the absence and presence of total and purified

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(Thaumatin-like proteins and chitinase) wine proteins. The results showed that in general bentonite

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alone has a low effect on the loss of terpenes, but removed ethyl esters and fatty acids. The presence

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of wine proteins in the solution treated with bentonite tended to increase the loss of esters with the

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longest carbon chains (from ethyl octanoate to ethyl decanoate), and this was significant when the

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purified proteins were used. The results here reported suggest that hydrophobicity can be one of the

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driving forces involved in the interaction of aromas with both bentonite and proteins.

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Keywords: wine aroma, bentonite, wine proteins, ethyl esters, terpenes

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INTRODUCTION

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The presence of haze in bottled white wines results in a serious quality defect because turbidity

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makes the wine undesirable for consumers. Wine proteins, which have the tendency to insolubilize

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during wine storage1-3, are the main cause for this defect. The majority of wine proteins derive from

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grapes, and, in particular, the haze-forming proteins have been found by several authors to be grape

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specific4-6. These proteins have been identified as grape pathogenesis related and, due to their

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resistance to proteolysis and their stability at acidic pH3, they are able to persist throughout the

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winemaking process.

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The addition of bentonite, a montmorillonite clay, is universally employed in the wine industry for

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the prevention of white wine protein hazing. Bentonite, which carries a net negative charge, interact

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electrostatically with the positively charged wine proteins, which results in their removal from the

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wine. However, bentonite action has been shown to be non specific for proteins, as it also removes

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other charged species or aggregates7 and also a direct interaction between bentonite and aroma

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compounds has been demonstrated in model solutions containing selected aromatic molecules8.

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This would contribute to the loss of sensorial quality that is often claimed for bentonite treated

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white wines.

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However in addition to the direct bentonite-aroma compounds interaction mechanism, the

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possibility that part of the aroma compound are removed because they are bound to the wine

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proteins cannot be excluded.

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Many other papers attempted to investigate bentonite-aroma interaction, but most of them were

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performed by headspace analysis or in model solution containing only some selected aroma

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molecules representative of different chemical classes9-11.

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In addition, the synergistic effect of proteins and bentonite in removing aroma compounds has been

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supposed by many authors10-12 but this phenomenon has been demonstrated only once13. In that case

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the effect on selected aromas (β-ionone and γ-decalactone) was, however, studied in a model



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solution in the absence of ethanol, with ovalbumin as standard protein and with a protein

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concentration ten times higher than that normally present in wine.

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A more detailed study of the interactions among aroma compounds, bentonite and wine proteins is

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then needed to understand the nature of mechanisms involved and to suggest the best practices for

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bentonite treatment in order to preserve as much as possible wine aroma. In order to assess the

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influence of wine proteins on the aroma composition of wines treated with bentonite, in this paper

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the effect of bentonite fining in a wine model solution containing the aroma compounds extracted

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from wine was studied in the absence and presence of total and purified wine proteins.


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MATERIALS AND METHODS

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Chemicals. Standards of 1-heptanol and 1-decanol were purchased from Carlo Erba Reagents

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(Milan, Italy). Ethyl decanoate and ethyl dodecanoate were purchased from Fluka (Sigma-Aldrich

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srl, Milan, Italy), ethyl hexanoate from B.H.D Laboratory Chemical Division (Poole, England),

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ethyl octanoate from Eastman Organic Chemicals (Rochester, US).

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Extraction of proteins and polysaccharides. For the extraction of wine macromolecules an

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untreated Manzoni Bianco wine (vintage 2011) produced in the Veneto region was used. This type

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of wine was chosen because it contains a high protein amount14. Macromolecules were purified

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following the preparative method proposed by Van Sluyter et al.15 with some modifications. Ten

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liters of wine were treated with 4 g/L PVPP (Sigma-Aldrich, Milan, Italy) and 1.5 g/L charcoal

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(Sigma-Aldrich, Milan, Italy) for 24 h at 15 °C in order to remove most of the polyphenols. The

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wine was then filtered with GF/A filters (Whatman, Kent, UK) and finally at 0.45 µm with cellulose

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acetate membranes (Sartorius AG, Göttingen, Germany) and brought at pH 3.0 with HCl. Aliquots

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of 5 liters were loaded at 8 mL/min on a S-Sepharose column (5 x 14 cm) (Pharmacia, Uppsala,

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Sweden) previously equilibrated in trisodium citrate buffer 30 mM pH 3.0. After washing with two

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column volumes with the same buffer, proteins were eluted with a gradient of NaCl from 0 to 1 M.

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Both the flow through (containing the unbound material, i. e. mainly polysaccharides) and the pool

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of the eluted peaks (representing the total proteins) were ultrafiltered at 3 kDa with an Amicon 8400

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apparatus (Sartorius), then dialyzed on regenerate cellulose membrane (3.5 kDa, Cellu-Sep®)

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against water and finally freeze dried. Five litres of the same wine were used for the purification of

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two main wine proteins (Chitinase and the Thaumatin-like protein VVTL1). In this case the

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fractions obtained after cation exchange separation on S-Sepharose were diluted in 1.25 M

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ammonium sulphate and further purified by Hydrophobic Interaction Chromatography (HIC) on a

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Phenyl-Sepharose HP column (GE-Healthcare) as follows. After washing the column with 1.25 M

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ammonium sulfate in 50 mM sodium citrate, pH 5.0, proteins were eluted with a 110 min linear 5 ACS Paragon Plus Environment


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gradient up to 100% 50 mM sodium citrate, pH 5.0. The peaks obtained with this second step were

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dialyzed against water (3.5 kDa MWCO), and freeze dried. The purity and identity of the fractions

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were assessed by RP-HPLC15 and SDS-PAGE16.

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Aroma extraction. Fermentative compounds and grape volatiles were extracted from a dry

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Moscato bianco wine (vintage 2011) produced in the Colli Euganei (Veneto, Italy) not previously

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treated with bentonite by using the method previously reported for wines and model wine

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solutions17. This type of wine was chosen because of its high content of primary aroma compounds.

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Aliquots of 500 mL were spiked with 4 mL of 1-heptanol (442 mg/L) as the internal standard, and

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extracted 3×50 mL with dichloromethane (Carlo Erba, Milano. Italy). Extraction was carried out

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under agitation for 15 minutes each time. The organic phase containing the aroma compounds was

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collected and washed 3×30 mL with 5% NaHCO3 to remove the acidic compounds by salification

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and shift into aqueous phase. After dehydration with anhydrous sodium sulfate, dichloromethane

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was filtered on blue ribbon filters 589/3 (Whatman, UK). The extract was concentrated first using a

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Vigreux column and finally under nitrogen flow. This extract was then used for the reconstitution

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experiments in model wine solution.

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Fifty mL of the clear supernatant of each reconstituted sample were used for the extraction of the

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aroma compounds remaining after bentonite treatment. The samples were spiked with 400 µL of 1-

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decanol 450 mg/L as the internal standard and extracted 3×15 mL with dichloromethane. After

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dehydration with anhydrous sodium sulfate and filtration as previously described, the extracts were

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concentrated with a Vigreux column (l=40 cm) to approximately 3 mL and adjusted to 1 mL under

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nitrogen flow before GC/MS analysis.

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Stability test. The purified proteins were suspended at 100 mg/L in model wine (tartaric acid 5 g/L,

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ethanol 12%, pH 3.2) or in a ultrafiltered Manzoni bianco wine (3.5 kDa). Bentonite Nucleobent

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(EVER, Pramaggiore, Italy) was prepared at 5% (w/v) in water for 24 h, and then different amounts

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(5, 10, 15 g/hL, final concentrations) were added to the wine protein solution. After 1 h at room

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temperature the solutions were filtered at 0.45 µm on cellulose acetate membrane (Sartorius). In 6 ACS Paragon Plus Environment

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order to assess the bentonite dose necessary to reach the protein stability, the protein solutions

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prepared in ultrafiltered wine were subjected to the heat test as reported by Pocock and Rankine18.

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Briefly, the samples were heated at 80°C for 6 h, followed by incubation at 4°C for 12 h, then the

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turbidity was measured with a nefelometer (HACH). When the difference in the turbidity measured

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before and after the heating was less than 2 NTU the sample could be considered stabile. For the

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samples prepared in model solution the dose of bentonite necessary for stabilization was instead

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calculated as the amount able to reduce the protein content below 20 mg/L.

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Determination of the protein content. The protein content was measured according to Vincenzi et

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al.19. Firstly, proteins were precipitated from 1 mL of wine with 10 µL of SDS 10% followed by

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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

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serum albumin (BSA, Sigma, Milan, Italy) in water.

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Electrophoresis. Electrophoretic analyses were performed according to Laemmli21. Samples to be

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analyzed were dissolved in a 0.5 M Tris-HCl pH 6.8 buffer containing 15% (v/v) glycerol (Sigma-

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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


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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

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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

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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

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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

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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)

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Spectral Database and the homemade CRA-VIT database ESTRATTI. For some compounds, the

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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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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

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P< 0.05 or P

Study of Combined Effect of Proteins and Bentonite Fining on the Wine

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