Research Note Use of Zirconium Dioxide during Fermentation as an Alternative to Protein Fining with Bentonite for White Wines

Transcription

1 Research Note Use of Zirconium Dioxide during Fermentation as an Alternative to Protein Fining with Bentonite for White Wines Marco Lucchetta, 1 Kenneth F. Pocock, 2 Elizabeth J. Waters, 2,3 and Matteo Marangon 2 * Abstract: Zirconia pellets (25 g/l) enclosed in a metallic cage were added on the second day to fermenting Riesling, Sauvignon blanc, and Semillon juices. After 48 hours, the zirconia-treated juices showed a large decrease in protein content and the resulting wines were heat stable. Compared to control juices, the fermentation rate was significantly increased for two juices and unchanged in the other juice. There were reductions in concentration of some mineral elements and tartaric acid and increases in ph in the resulting wines from the zirconia-treated juices. Key words: fermentation, haze, juice, protein stability, wine, zirconium dioxide 1 Department of Agronomy, Food, Natural Resources, Animals, and Environment, Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia, University of Padua, via dell Università 16, Legnaro, Italy; 2 The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia; and 3 Grape and Wine Research and Development Corporation, P.O. Box 610, Kent Town, SA 5071, Australia. *Corresponding author ( matteo.marangon@awri.com.au; tel: ; fax: ) Acknowledgments: This work was supported financially by Australia s grapegrowers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. The AWRI is a member of the Wine Innovation Cluster located in Adelaide. The work of M. Lucchetta is part of the research activity of the Dottorato di Ricerca in Viticoltura, Enologia e Marketing delle imprese vitivinicole supported by Provincia di Treviso (Italy) and it has been supported in part by the Fondazione Ing. Aldo Gini. The authors thank Paul Smith, AWRI, for helpful discussion regarding this work, and Francisco López, Universitat Rovira i Virgili (Spain), for donating the zirconia. Publication costs of this article defrayed in part by page fees. Manuscript submitted Dec 2012, revised Apr 2013, accepted May 2013 Copyright 2013 by the American Society for Enology and Viticulture. All rights reserved. doi: /ajev Residual grape proteins present in bottled wines can unfold and aggregate during storage to form a haze (Bayly and Berg 1967, Waters et al. 2005). Hazy wines are perceived as faulty by consumers. Thus, thaumatin-like proteins and chitinases, the proteins responsible for wine hazing (Waters et al. 1996), need to be removed and bentonite fining is currently used to remove them. Bentonite is a clay cation exchanger that, after binding the wine proteins, settles to the bottom of the tanks. Bentonite is still extensively used because it is relatively inexpensive and quite effective in protein removal, even if its use has many indirect costs and can negatively affect the sensorial profile of the treated wines (Waters et al. 2005). In the last 25 years researchers have tried to find alternatives to bentonite (Hsu et al. 1987, Powers et al. 1988, Waters et al. 1992, Sarmento et al. 2000, Vincenzi et al. 2005, Marangon et al. 2012a, 2012b), but to date no other method has been adopted by the wine industry. Among the alternatives proposed is zirconium dioxide (zirconia), a metal oxide able to adsorb the unstable wine proteins. Pachova et al. (2002) were the first to propose the use of zirconia in enology and to prove that this material could adsorb heat unstable proteins to stabilize white wines. In further work, zirconia was used in a continuous system (Pashova et al. 2004) and zirconia treatments have been compared against bentonite fining (Salazar et al. 2006). Despite being promising, zirconia treatment has not been adopted by the wine industry. An efficient continuous process is not possible because of the slow protein adsorption rate and a difficult regeneration procedure. In previous work (Marangon et al. 2011), we addressed some of these issues with the proposal of a discontinuous process in which zirconia was enclosed in a metallic cage and left in contact with wine for the time required to reach protein stability. With this approach we obtained heat-stable wines with no losses as lees, since zirconia pellets could simply be removed and regenerated several times by an easy washing procedure, thus reducing the costs of its application. Although results of this work were encouraging, one requirement of successful protein removal was that the wine had to be mixed constantly for 3 to 5 days, an expensive operation to perform in a winery. A possible solution would exploit the natural mixing occurring during fermentation. The aim of this project was to determine the effectiveness of zirconia when added during fermentation in removing proteins, including its effect on fermentation rate and on the chemical parameters of the resulting wines. Materials and Methods Materials. Three unfined 2007 juices (Riesling, Sauvignon blanc, and Semillon) sourced from Adelaide Hills (South Australia) were inoculated with Saccharomyces cerevisiae AWRI 796 yeast (Maurivin, Toowoomba, QLD, Australia). The zirconia was in pellet form (Saint-Gobain, 400

2 Protein Stabilization with Zirconia during Fermentation 401 Akron, OH) and was donated by Prof. Francisco Lopéz. The pellets consisted of 3 mm diameter disks with 1 mm thickness, 6.2 nm pore size, m 2 /g surface area, with a tetragonal morphology (Salazar 2007). Enological analyses. Conventional enological analyses were conducted by the Commercial Service of The Australian Wine Research Institute using standard methods. Alcohol, specific gravity, ph, titratable acidity, glucose/fructose, and volatile acidity analysis were measured using a WineScan FT 120 as described by the manufacturer (Foss, Hillerød, Denmark). Free and total SO 2 were measured by the aspiration method (Rankine and Pocock 1970). Soluble solids were measured by refractometry. Haze formed upon heat test was measured as indicated by Pocock and Waters (2006). SDS PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed with NuPage 4 12% Bis-tris gels (Invitrogen, Carlsbad, CA) 1.5 mm thick, 15 wells) using an XCell SureLock Mini Cell (Invitrogen) as described previously (Marangon et al. 2011). Protein content, organic acids, and elemental analysis. Protein content was determined by EZQ protein quantification kit (Invitrogen), as described previously (Marangon et al. 2011). The concentration of organic acids (citric, tartaric, malic, succinic, and lactic) was determined by HPLC as described previously (Marangon et al. 2011). Elemental composition was determined by inductively coupled plasma optical emission spectrometry by Waite Analytical Services (University of Adelaide, Glen Osmond, Australia). Experimental design. The three unfined juices were fermented in triplicate in 250 ml Schott bottles (150 ml juice volume) in the presence or absence of 25 g/l zirconia. Zirconia pellets were enclosed in a handmade stainless-steel infuser ball of ~2 cm diameter and added to juice samples. Control wines were produced using an empty infuser. The infusers were suspended in the juices at day 2 from the beginning of fermentation and left in contact with juices for three days. Fermentations were conducted at 18 C. After yeast inoculation, the fermentation rate was monitored daily by refractometric analysis. An addition of 300 mg/l diammonium phosphate was made to all fermentations at day 3 (approximately halfway through the fermentations). In the latter stages of the fermentations, reducing sugars were estimated by Foss WineScan and the fermentations were considered finished when glucose/fructose was <1 g/l. Finished wines were stored at 4 C for five days before being sterile filtered (0.2 µm) and 40 mg/l potassium metabisulfite was added to prevent oxidation. Each of the six fermentations was done in triplicate, and analyses for each replicate were performed in triplicate unless otherwise stated. Statistical analysis. Data were analyzed by one-way completely randomized ANOVA with JMP Statistical Discovery Software (ver a; SAS Institute, Cary, NC) and data significance assessed by Student s t test. Results and Discussion The general composition of the six wines produced is shown (Table 1). All three wines from zirconia-treated juices had reduced alcohol content, specific gravity, and titratable acidity and increased ph. The diminution in titratable acidity was mostly explained by the significant reduction in tartaric acid for the three wines (relative reduction by 10 to 18%) (Table 2). Citric acid content was significantly decreased in Table 1 Enological analysis of the three wines produced without (Control) or with (Zirconia) 25 g/l zirconia. Data are from single replicates. Riesling Sauvignon blanc Semillon Parameter Control Zirconia Control Zirconia Control Zirconia Alcohol (% v/v) Specific gravity ph Titratable acid to ph Glucose + fructose Volatile acidity as acetic acid Sulfur dioxide free/total (mg/l) <4/47 <4/44 <4/27 <4/22 <4/38 <4/41 Table 2 Organic acid concentration of wines after treatment with zirconia. Each data point represents the average of at least three independently prepared samples. Means followed by a different letter are significantly different (p < 0.05) according to Student s t test. Wine/ treatment Citric Tartaric Malic Succinic Lactic Total acidity a Riesling Control 0.16 a 3.50 a 2.38 b 4.07 a 0.37 a a 25 g/l Zirconia 0.13 b 3.10 b 2.50 a 3.96 a 0.35 a a Sauvignon blanc Control 0.18 a 3.74 a 2.29 a 2.61 a 0.17 a 8.99 a 25 g/l Zirconia 0.13 b 3.08 b 2.17 b 2.65 a 0.19 a 8.22 a Semillon Control 0.12 a 4.45 a 2.21 a 1.82 a 0.22 a 8.82 a 25 g/l Zirconia 0.09 b 4.00 b 2.12 a 1.65 b 0.18 a 8.05 b a Total acidity is the sum of the five acids.

3 402 Lucchetta et al. the three wines as well (relative reduction by 19 to 28%), while the other organic acids were mostly unaffected. Since it has been demonstrated previously that zirconia application to finished wines decreases the content of several elements (Marangon et al. 2011), the effect of its use in fermentation was assessed (Table 3). In general, all the wines produced with zirconia contained significantly less K, P, S, Mn, and B, while only two wines produced with zirconia contained less Ca, Cu, and Fe. The only element with a significantly increased content after zirconia treatments in all the wines was Na, while the possible presence of residual zirconia in treated wines could not be measured because of limitations of the method used. In general, these trends are consistent with previous findings (Marangon et al. 2011). As the ability of zirconia in removing some elements could have some impact in the fermentation performance of yeast (Sablayrolles 2009), the fermentation rates were monitored daily (Figure 1). The presence of zirconia significantly increased the rate of fermentation for two of the three juices, while the other was unaffected, possibly because zirconia: (1) binds a small portion of sugar, thus explaining the lower alcohol content of zirconia treated wines; (2) acts as support for the yeast; (3) removes some ions, such as copper, which are detrimental to yeast activity (Ferreira et al. 2006). The protein content was monitored daily during the fermentation (Figure 2). The effect of zirconia addition on protein removal is evident, as the addition of the adsorbent at day 2 corresponded to a sharp decrease in soluble proteins for each of the three wines. This large decrease was observed for two days (days 3 and 4), while at the third day of application (day 5) most of the proteins were already removed and therefore the rate of removal was slower. At the end of fermentation each of the zirconia-treated wines contained minimal proteins (5 to 20 mg/l), amounts ~90% lower than those of the control wines. At this addition rate, applying zirconia for two days was sufficient to remove the majority of the proteins (Figure 2). Figure 1 Fermentation rate in Riesling, Sauvignon blanc, and Semillon measured daily by refractometer. Each data point represents the average of at least three independently prepared samples. Within each time point, measurements with different letters are significantly different (p < 0.01) according to Student s t test. Table 3 Elemental composition of wines after treatment with zirconia (25 g/l). Each data point represents the average of at least three independently prepared samples. Means followed by a different letter are significantly different (p < 0.05) according to Student s t test. Element a (mg/l) Riesling Semillon Sauvignon blanc Control Zirconia Control Zirconia Control Zirconia K a b a b a b Ca a b a b a a Mg a b a a a a P a b a b a b S a b a b a b Na b a b a b a Mn 0.28 a 0.19 b 1.01 a 0.68 b 0.70 a 0.61 b B 4.48 a 4.03 b 7.72 a 7.08 b 5.95 a 5.25 b Cu <0.03 a <0.03 a 0.04 a <0.03 b 0.11 a <0.03 b Zn 0.03 b 0.08 a 0.04 a <0.02 b 0.22 a 0.16 a Al 0.04 a <0.01 b 0.03 a 0.04 a 0.08 a 0.11 a Fe 0.19 a 0.01 b 0.05 a <0.02 b <0.02 a 0.04 a a Mo, Co, Ni, Cr, Cd, Pb, and Se were not included in the table because their contents were below the detection limit (0.06, 0.06, 0.07, 0.04, 0.02, 0.2, and 0.6 mg/l, respectively).

4 Protein Stabilization with Zirconia during Fermentation 403 Figure 2 Kinetics of protein removal from the three juices during fermentation. Zirconia (25 g/l) was added at day 2 and removed at day 5. The protein removal was assessed qualitatively via SDS- PAGE analysis (Figure 3) and RP HPLC analysis (data not shown). The gels highlighted the nonspecific protein removal by zirconia. There were only traces of protein bands left in the zirconia-treated wines in each of the three gels. In particular, the proteins responsible for haze formation, thaumatin-like proteins (bands at 23 kda) and chitinases (bands at 25 kda), were almost fully removed by zirconia for each of the wines. Despite the observed protein reduction, an essential step for assessing the efficacy of protein fining is to challenge the treated wines by a heat test and to measure the haze formed (Figure 4). Each zirconia-treated wine passed the heat test (net haze <2 NTU), indicating that they were fully stabilized by the treatment. A previous work demonstrated that the application of 25 g/l zirconia could fully or partly stabilize finished wines after a minimum contact time of 72 hr (Marangon et al. 2011). Therefore, the application of zirconia during fermentation resulted in a more effective and rapid removal of proteins. Preliminary trials indicate that the dosage of zirconia applied Figure 3 The effect of zirconia treatments on the protein composition of the three wines shown by SDS-PAGE. Proteins from 100 µl wine were loaded per lane; three replicates made for each of the treatments are shown. Juice: juice before the beginning of fermentation; zirconia: wines treated with 25 g/l zirconia; control: wines obtained without zirconia. Figure 4 Haze formed upon heat test of the three wines obtained from the fermentation of juices with (zirconia) or without (control) 25 g/l zirconia (n = 3).

5 404 Lucchetta et al. during fermentation could be reduced from the amount used in this work, which would result in a more economical process. However, since in this work protein removal was almost complete with two days of contact, it is likely that addition of lower dosages for longer times would represent a more feasible solution. Conclusions Zirconia in pellet form enclosed in a metallic cage is a viable alternative to bentonite fining for protein removal from juice and wine. Its application during fermentation is an improvement from a previously proposed use of zirconia in wine, since stirring is replaced by natural mixing during fermentation. Other key advantages of using zirconia in fermentation are that the fermentation rate is increased, that it is likely that the dosage can be reduced, and most importantly, that the wines produced are fully heat stable with no loss of wine as lees, since the cage with the pellets can simply be removed and regenerated several times with a previously proposed washing procedure. Adding zirconia during fermentation, thus avoiding prolonged stirring, is potentially less costly than the previously proposed application on finished wines. However, the status of zirconia dioxide as an allowed winemaking additive remains to be established and should be determined by the practitioner planning to use the material. The possible presence of residual zirconia in treated wines also warrants further investigation. Literature Cited Bayly, F.C., and H.W. Berg Grape and wine proteins of white wine varietals. Am. J. Enol. Vitic. 18: Ferreira, J., M. Du Toit, and W.J. Du Toit The effects of copper and high sugar concentrations on growth, fermentation efficiency and volatile acidity production of different commercial wine yeast strains. Aust. J. Grape Wine Res. 12: Hsu, J.C., D.A. Heatherbell, J.H. Flores, and B.T. Watson Heatunstable proteins in grape juice and wine. II. Characterization and removal by ultrafiltration. Am. J. Enol. Vitic. 38: Marangon, M., M. Lucchetta, and E.J. Waters Protein stabilisation of white wines using zirconium dioxide enclosed in a metallic cage. Aust. J. Grape Wine Res. 17: Marangon, M., M. Lucchetta, D. Duan, V.J. Stockdale, A. Hart, P.J. Rogers, and E.J. Waters. 2012a. Protein removal from a Chardonnay juice by addition of carrageenan and pectin. Aust. J. Grape Wine Res. 18: Marangon, M., S.C. Van Sluyter, E.M.C. Robinson, R. Muhlack, H. Holt, P.A. Haynes, P.W. Godden, P.A. Smith, and E.J. Waters. 2012b. Degradation of white wine haze proteins by Aspergillopepsin I and II during juice flash pasteurization. Food Chem. 135: Pachova, V., M. Ferrando, C. Güell, and F. López Protein adsorption onto metal oxide materials in white wine model system. J. Food Sci. 67: Pashova, V., C. Güell, E. Pueyo, M. López-Barajas, M.C. Polo, and F. López White wine protein stabilization by a continuous process using a packed column. Am. J. Enol. Vitic. 55: Pocock, K.F., and E.J. Waters Protein haze in bottled white wines: How well do stability tests and bentonite fining trials predict haze formation during storage and transport? Aust. J. Grape Wine Res. 12: Powers, J.R., C.W. Nagel, and K. Weller Protein removal from a wine by immobilized grape proanthocyanidins. Am. J. Enol. Vitic. 39: Rankine, B.C., and K.F. Pocock Alkalimetric determination of sulphur dioxide in wine. Aust. Wine Brew. Spirit Rev. 88: Sablayrolles, J.M Control of alcoholic fermentation in winemaking: Current situation and prospect. Food Res. Int. 42: Salazar, F.N White wine continuous protein stabilization: Industrial viability. PhD thesis, Universitat Rovira i Virgili, Tarragona, Spain. Salazar, F.N., I. Achaerandio, M.A. Labbe, C. Güell, and F. López Comparative study of protein stabilization in white wine using zirconia and bentonite: Physicochemical and wine sensory analysis. J. Agric. Food Chem. 54: Sarmento, M.R., J.C. Oliveira, and R.B. Boulton Selection of low swelling materials for protein adsorption from white wines. Int. J. Food Sci. Tech. 35: Vincenzi, S., M. Polesani, and A. Curioni Removal of specific protein components by chitin enhances protein stability in a white wine. Am. J. Enol. Vitic. 56: Waters, E.J., G. Alexander, R. Muhlack, K.F. Pocock, C. Colby, B.K. O Neill, P.B. Hoj, and P. Jones Preventing protein haze in bottled white wine. Aust. J. Grape Wine Res. 11: Waters, E.J., N.J. Shirley, and P.J. Williams Nuisance proteins of wine are grape pathogenesis-related proteins. J. Agric. Food Chem. 44:3-5. Waters, E.J., W. Wallace, and P.J. Williams Identification of heat-unstable wine proteins and their resistance to peptidases. J. Agric. Food Chem. 40: