Managing wine extraction, retention, clarity and stability for defined styles and efficient production
The project will investigate:
- the role of macromolecules such as tannins, polysaccharides, proteins and their aggregate colloids, and their impact on stability, clarity, filtration and fouling;
- the impact of other wine matrix components on macromolecule extraction, retention and function;
- the source of these molecules or their precursors in grapes and yeast, and the impact of winemaking processes such as clarification, flotation, vinification and filtration on their retention and/or transformation;
- the impact of fouling of surfaces by macromolecules leading to production inefficiencies;
- alternative strategies for achieving protein stability and cold stability, for example, through use of novel additives and/or processing techniques;
- practical methods for wineries to determine likely extractability of macromolecules during winemaking and the factors that affect extraction and retention (e.g. enzymes, water additions and heat treatments).
Using Fluorescence Correlation Spectroscopy to understand how wine macromolecules interact
Despite the large body of literature focused on interactions between polysaccharides, proteins and phenolic compounds (tannins), a great deal of information is still lacking about the unique medium of wine, especially regarding the structure of complexes at the molecular and macromolecular level. In part, this can be attributed to limitations of current analytical techniques. Currently, it is difficult to identify experimental methods that are sufficiently sensitive to discriminate among the structures of wine macromolecules without disturbing the complex wine matrix. In a collaborative project with the Max Planck Institute for Polymer Research in Germany, this knowledge gap was addressed by employing Fluorescence Correlation Spectroscopy (FCS) for the first time in wine research (Figure 12). This technique offers an opportunity to study molecular and macromolecular aggregation by the addition of only tiny amounts of fluorescently labelled molecules of interest (such as proteins, polysaccharides or phenolic compounds) and therefore allows visualisation without disturbing the wine matrix.
Bovine serum albumin (BSA) was used as a model protein due to its structural similarity to grape and salivary proteins, both of which are relevant to wine colloidal systems. The structural changes and conformational dynamics of BSA induced by ethanol, polysaccharides and tannins were investigated. Experiments primarily focused on the impact of the following aspects on protein aggregation:
- ethanol concentration
- grape skin and seed tannin
- a mixed macromolecule matrix.
Conformational relaxation time components of native BSA drastically varied with the addition of wine macromolecules, signifying changes in conformation dynamics. The effect of tannin type (seed vs skin) on protein aggregation was very significant, pointing to a higher affinity of BSA towards seed tannin. The effect of wine polysaccharides was also very pronounced, with a rhamnogalacturonan II-enriched fraction inducing protein aggregation, and a mannoprotein-enriched fraction having the opposite effect. This work demonstrated that FCS holds promise for investigation of interactions between wine macromolecules in model and real wine systems.
New analytical methods to help manage heat and cold stability
Two new methods have been developed to assist with management of wine instabilities, one to determine the concentration of heat-unstable proteins in white wines using a fluorescent dye and a second to accurately quantify tartrate crystal formation after a three-day cold test. It is hoped that the new test for heat-unstable proteins will enable wine¬makers to easily and accurately determine if their wines are above or below a haze formation threshold for protein concentration, as it relates to the existing heat test. The new cold stability test subjects a wine to the same conditions as the standard three-day test, but instead of observing crystal formation with the naked eye, recovers and quantifies the crystals formed. To validate the method, ten unstable wines were analysed using the new approach. Results were compared to those from the standard three-day test and a mini-contact (conductivity) test and were well correlated for all wines. Future work will use the newly developed method to assess the role of wine macromolecules in the development of cold instability.
Investigating zeolite’s effect on cold stability
In early trials of zeolite as an alternative to bentonite for heat stabilisation of wines, it was interesting to note that the concentration of potassium in wines decreased by more than 30% following zeolite treatment. This suggested that zeolite addition might also be a viable approach to improve cold stability in wine and this was investigated during the year. Encouraging preliminary results showed that zeolite treatment brought three cold-unstable white wines to, or very close to, cold stability as measured using the mini-contact test. This test measures the change in conductivity over time of a wine that has been chilled, mixed and heavily super-saturated with powdered KHT. A result of less than 5% change in conductivity indicates a cold-stable wine. The stabilising effect of zeolite was observed in a Muscat Gordo wine immediately following zeolite treatment, with a conductivity change below 2%, and for the Sauvignon Blanc and Chardonnay wines a progressive crystallisation of KHT was observed at 15°C over four months (Figure 13A), with conductivity changes of 5 and 5.5%, respectively. This suggests that zeolite may not adsorb potassium, but rather may help initiate crystallisation, inducing the loss of potassium from wine as KHT. Importantly, results indicated that zeolite could simultaneously heat and cold stabilise wines. Moreover, with precipitation of KHT crystals occurring at 15°C, zeolite use could potentially remove the need to chill wine as part of the cold stabilisation process, saving significant amounts of energy. As depicted in Figure 13B, reductions in potassium content of 40%, 20% and 30% were observed for the zeolite-treated Muscat Gordo, Chardonnay and Sauvignon Blanc wines, respectively. A further positive outcome was that despite losses in tartaric acid due to crystallisation, changes in wine pH were negligible for the Sauvignon Blanc and Chardonnay wines and small for the Muscat Gordo wine (Figure 13C), and thus substantial doses of tartaric acid may not be required after treatment. Work to understand the sensory impacts of zeolite addition on white wines is being finalised.
Proteins in red wine – are they important?
Wine protein is most commonly considered in relation to white and rosé wines, since these are the wines most susceptible to the development of visible protein haze. In red wine, protein is often thought to be absent, being removed during the early stages of winemaking due to precipitation of tannin-protein complexes. Earlier research at the AWRI revealed that in red grapes, protein is present in sufficient quantities to precipitate a substantial amount of tannin that would otherwise be extracted into wine (Bindon et al. 2016). Furthermore, in cold-hardy hybrid (non-vinifera) grape varieties that contain higher levels of protein than Vitis vinifera grapes, high concentrations of residual protein remain in wine and can precipitate oenological tannins added during winemaking (Springer et al. 2016). Based on these observations, it was of interest to understand how important protein is in modifying macromolecule extraction and stability in red wines.
Initially, existing methods for protein analysis were used, but they were found to be unsuitable for red wine due to interference from wine tannins. A new method was developed and validated specifically for protein quantification in red wines, and more than 100 red wines, including wines made from non-vinifera varieties and their interspecific hybrids sourced from the USA, were analysed. Protein concentrations in Australian Vitis vinifera red wines ranged from 16 mg/L to 113 mg/L with a median of around 50 mg/L (Figure 14). Protein concentration was found not to vary according to grape variety in Vitis vinifera red wines. Wines made from non-vinifera varieties and their interspecific hybrids had a wide range of protein concentrations from as low as 13 mg/L up to 350 mg/L, with a median of 70 mg/L. It was noteworthy that the cold-hardy hybrid varieties had the highest protein concentrations, with a median of 124 mg/L.
Given the relevance of protein to the development of haze in white wine, it was considered that high-protein red wines might in fact be heat-unstable, and potentially present difficulties with filtration or develop deposits following addition of oenotannin or during ageing. This was particularly relevant, given that red wines could contain protein concentrations at levels that would result in haze in white wines. It was found that while most red wines (both vinifera and non-vinifera varieties) were not heat stable according to a red wine heat test, the extent of heat instability did not increase with increasing protein concentration. While it was also found that unfiltered wines did not necessarily have higher protein concentration than filtered wines, the role of red wine proteins in membrane fouling during filtration will continue to be studied. Although bentonite treatment of commercial USA hybrid wines known to be influenced by excess protein was not able to remove protein, experiments are underway to determine whether protein can be enzymatically broken down during fermentation. It is thought that by uncoupling protein-tannin interactions during fermentation, wine tannin concentration may be improved in low-tannin grape varieties, such as Pinot Noir.
Bindon, K.A., Li, S., Kassara, S., Smith, P.A. 2016. Retention of proanthocyanidin in wine-like solution is conferred by a dynamic interaction between soluble and insoluble grape cell wall components. J. Agric. Food Chem. 64(44): 8406-8419.
Springer, L.F., Sherwood, R.W. Sacks, G.L. 2016. Pathogenesis-related proteins limit the retention of condensed tannin additions to red wines. J. Agric. Food Chem. 64(6): 1309-1317.