MARANGON, MATTEO

Smith, P. A.

English

Archivio istituzionale della ricerca - Università di Padova

Clarifying the mechanism of protein haze formation in white wines Matteo Marangona,b, Paul A. Smithba The Australian Wine Research Institute, P.O. Box 197, Glen Osmond (Adelaide), SA 5064, Australia.b Current address: Wine Department, Plumpton College, Ditchling Road, Plumpton, Nr Lewes, East Sussex BN7 3AE, UK. Keywords: wine; protein; haze; mechanism.1. INTRODUCTION Protein haze formation in white wines remains one of the key potential instabilities in wine production that requires costly treatment with losses in overall quality. Haze formation is a serious quality defect because consumers perceive hazy wines as faulty. This problem is due to the presence of residual grape pathogenesis-related (PR) proteins in wines after bottling, mainly thaumatin-like proteins (TLPs) and chitinases [1]. These are haze-forming proteins that can become unstable and unfold, an occurrence favoured by exposure of wine to high temperatures (eg during transport, fast aggregation) or inappropriate storage conditions (slower kinetic of aggregation). Once the proteins are in unfolded status they can aggregate into light-dispersing particles, a step that can be favoured by non-proteinaceous compounds present in the wine, to make wines appear turbid [1]. Since hazy wines are not saleable, haze appearance is generally prevented via bentonite fining, a process effective in removing the grape proteins, but with several drawbacks such as wine volume loss and disposal costs, as well as detrimental effects on wine flavour and quality and undesirable processing complexity [2]. Hence, alternatives are sought, but so far the search for alternatives to bentonite has not yet resulted in commercially viable solutions able to compete with bentonite's efficacy and low cost.It is believed that an in depth understanding of the mechanisms of protein haze formation is needed, because it has the potential to lead to the development of novel, efficient, and environmentally sustainable winemaking processes to prevent haze from forming. In this article an overview of the recent advances obtained by our group on the study of protein instability of white wines in given, including: i) notions on the role of purified wine proteins, ii) explanation of the role of some non-proteinaceous wine components, iii) notions on the effect of different unfolding temperatures and unfolding/aggregation behaviour of grape proteins. A revisited mechanism of haze formation accounting for the several recent breakthroughs in the field will be presented. 2. MATERIALS AND METHODS Purification of Proteins. Chitinases and TLPs were isolated from different unfined grape juices sourced from South Australia by strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) as described by Van Sluyter et al. [3]. Dynamic Light Scattering (DLS) measurement. DLS experiments were performed using a Malvern Zetasizer 3000 HS apparatus as described previously [4].Differential Scannig Calorimetry (DSC). Differential scanning calorimetry was undertaken with a VP-DSC apparatus as described previously [5].Scanning Ion Occlusion Sensing. The IZON qNano device was used to measure size and concentration of protein aggregates [6].Experimental approach. In order to study single variables involved in the mechanism of wine hazing, juices or wines were normally fractionated in their single components as proteins, polysaccharides, and phenolics. Purified wine components where then used as ‘ingredients’ and added back to the stripped wine or to a model wine at different concentrations, ratios and combinations. Protein aggregation was induced by heating/cooling cycles, and resulting aggregates measured via DLS, SIOS, or nephelometry [4‒7].3. RESULTS AND DISCUSSIONIn recent years our knowledge about the haze forming proteins improved significantly, particularly after the development of an efficient method for protein purification from juices and wines [3]. This fact allowed us to use wine fractionation and reconstitution experiments in order to explain in-depth the steps involved in the mechanism of haze formation, with particular attention to the role played by single proteins, polysaccharides, phenolics, temperature, ionic strength and sulfate. In one of these studies, the roles of wine ionic strength and presence of sulfate were investigated by DLS [4]. Sulfate concentration and the overall ionic strength of wines were found to play an important role on heat-induced formation of haze by purified TLPs and chitinases, with chitinases resulting more prone to aggregate than TLPs in the normal wine ionic strength range. In addition, sulfate was confirmed to be a primary actor in haze formation, likely by converting soluble aggregates into larger visible haze particles [4]. In other studies the purified proteins were analysed by DSC to assess their unfolding temperatures and behaviour [5]. Results indicated that temperature plays a key role for the onset of protein aggregation, and differences in the unfolding temperature of chitinases (approximately 55°C) and TLPs (approximately 62°C) were detected (Table 1). Significant differences in the proteins unfolding behaviour were also noted, with chitinases being unable to refold upon cooling (irreversible unfolding), while TLPs can (reversible unfolding) [5]. Table 1. Unfolding temperatures and reversibility behaviour of purified grape proteins.Protein	Unfolding temperature	Reversibility upon coolingChitinases	55°C	IrreversibleTLPs	56-62°C	ReversibleΒ-glucanases	<40°C	IrreversibleInvertases	81°C	IrreversibleLipid Transfer Proteins	>80°C	Not assessedIn another work, SIOS was used to measure the size and concentration of the aggregates formed upon heating/cooling cycles by purified proteins in reconstituted wines [6]. The primary role of chitinases in haze formation was again confirmed, since this class of proteins was found to be more reactive with other wine macromolecules than TLPs, to form bigger aggregates and to be more prone to precipitate. Therefore chitinases is considered as the major player in heat-induced haze in wines, even if other experiments proved that some TLPs isoform can form haze as well [6].As results of these recent breakthroughs we have reached a much deeper understanding of wine proteins and why they unfold and aggregate - findings that were used to elaborate a revisited ‘life cycle’ of proteins in wine (Figure 1). Figure 1. Mechanism of haze formation revisited: ‘life cycle’ of proteins in wine.After bottling, proteins in soluble status are found in wines in their folded 3D structure (Figure 1A). However, this folded structure can be lost (unfolding), a step that is commonly mediated by inappropriate storage temperatures. As found by DSC experiments, different proteins have different unfolding temperatures that for the haze-forming proteins is generally between 55-62ºC [5]. Moreover, DLS experiments showed that the wine matrix can play an important role for the onset of aggregation as high ionic strength can screen the proteins surface’s charges thus reducing proteins’ repulsion and favouring their aggregation [4]. Once the protein is unfolded (Figure 1B), it exposes its hydrophobic binding sites that are generally buried in the core of the protein. Protein aggregation then occurs by hydrophobic attractions with other unfolded proteins or with other wine components as phenolics (Figure 1C). With time, the protein aggregates can grow and cross-link with other aggregates, a step favoured by the presence of sulfate, phenolics and high ionic strength, until they reach a size that makes them visible to the naked eye (>1 µm) (Figure 1D).The increased level of understanding of the mechanism of haze formation places us in an excellent position for setting up better tools to predict haze potential of wines and for studying targeted alternatives to bentonite to prevent haze from forming in bottled white wines. For example, the notion that haze-forming proteins unfold between 55 and 62ºC was fundamental to develop a wine stabilisation strategy based on juice flash pasteurisation in presence of an enzyme, which showed the potential to eliminate the need for bentonite fining [8].References1.	E.J. Waters, G. Alexander, R.A. Muhlack, K.F. Pocock, C. Colby, B.K. O’Neill, P.B. Høj, P. Jones. Aust. J. Grape Wine Res. 11 (2) (2005) 215.2.	P. Majewski, A. Barbalet, E.J. Waters. Aust. N. Z. Grapegrower Winemaker 569 (2011) 58.3.	S.C. Van Sluyter, M. Marangon, S.D. Stranks, K.A. Neilson, Y. Hayasaka, Y., P.A. Haynes, R.I. Menz, E.J. Waters. J. Agric. Food Chem. 57 (23) (2009) 11376. 4.	M. Marangon, F.-X. Sauvage, E.J. Waters, A. Vernhet. J. Agric. Food Chem. 59 (6) (2011) 2652. 5.	R.J. Falconer, M. Marangon, S.C. Van Sluyter, K.A. Neilson, C. Chan, E.J. Waters. J. Agric. Food Chem. 58 (2) (2010) 975.6.	D. Gazzola, S.C. Van Sluyter, A. Curioni, E.J. Waters, M. Marangon. J. Agric. Food Chem. 60 (42) (2012) 10666.7.	M. Marangon, S.C. Van Sluyter, K.A. Neilson, C. Chan, P.A. Haynes, E.J. Waters, R.J. Falconer. J. Agric. Food Chem. 59 (2) (2011) 733.8.	M. Marangon, S.C. Van Sluyter, E.M.C. Robinson, R.A. Muhlack, H.E. Holt, P.A. Haynes, P.W. Godden, P.A. Smith, E.J. Waters. Food Chem. 135 (3) (2012) 1157.			

Clarifying the mechanism of protein haze formation in white wines

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Clarifying the mechanism of protein haze formation in white wines

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