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The Australian Wine Research Institute Recent development in removing protein haze from white wines Dr Jacqui McRae Research Scientist AWRI Team Texture jacqui.mcrae@awri.com.au

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The Australian Wine Research Institute Wine Haze

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The Australian Wine Research Institute Wine Haze is caused by Protein aggregation... With sulfate + Polysaccharides + Phenolics + Organic acids...and more...!

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The Australian Wine Research Institute Grape Invertase Glucanase Chitinases Thaumatin-Like Proteins Lipid Transfer Proteins Thaumatin-Like Proteins Grape Pathogenesis- Related (PR) proteins Haze-forming proteins Grape proteins that are resistant to winemaking

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The Australian Wine Research Institute 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 control1-510-30>30 Percent of powdery mildew infection Wine haze after heat test (Abs 540) Grape Pathogenis-Related (PR) proteins increase in concentration with infection Girbau, Stummer, Pocock, Baldock Scott and Waters, AJGWR 2004 Haze-forming proteins

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The Australian Wine Research Institute Bentonite CRAZY

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The Australian Wine Research Institute Bentonite: the good Cheap Readily available Easy to use Non-toxic The good Effective mechanism:

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The Australian Wine Research Institute Bentonite: the bad X Non-selective X Extra processing steps: water addition settling time waste removal X Bad press...? Wine taste funny? It may contain cat litter... Malcolm Gluck reveals the unpalatable truth about the wine industry Updated: 02:28 AEST, 28 September 2008 http://www.dailymail.co.uk/news/article-1063029/Wine-taste-funny-It-contain-cat-litter--Malcolm-Gluck-reveals-unpalatable-truth-wine- industry.html

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The Australian Wine Research Institute Bentonite: the bad $1 Billion wasted worldwide Majewski, P.; Barbalet, A.; Waters, E. J. The Australian & New Zealand Grapegrower & Winemaker 2011, 5862.

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The Australian Wine Research Institute Bentonite: making the most of it Inline dosing with centrifugation Proof of concept Wine storage tank Centrifuge Bentonite slurry Contacting Zone- pipe work from heat exchanger Static mixer Sight glasses Clarified wine Sludge Muhlack et al (2006) Aust. J. Grape Wine Res. 12, 221-234 Injection point

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The Australian Wine Research Institute WINE: 100 KL Bentonite Slurry: 1.5 kL FINED WINE: 96.4 KL ~3% Loss Low value wine: 4.6 kL Sludge: 0.4 kL (+ pearlite) (a) Batch fining Bentonite Slurry: 1.5 kL Sludge: 0.4 kL FINED WINE: 101 KL No loss No extra steps (c) In-line dosing with zero carry over Lees: 5 % Bentonite Slurry: 1.5 kL Sludge: 0.3 kL FINED WINE: 99.9 KL Double process steps Low value wine: 1.1 kL Sludge: 0.1 kL (+ pearlite) (b) In-line dosing with 30 % carry over Lees: 1.5 % Bentonite: making the most of it

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The Australian Wine Research Institute Criteria for bentonite alternatives: Better settling less waste More efficient use less material More selective Cost effective No flavour taint Bentonite: the crazy alternatives

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The Australian Wine Research Institute Bromelain Chitin Adsorbent resins Phytic acid Carrageenan Zirconia Bentonite: the crazy alternatives

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The Australian Wine Research Institute Zirconia - Fast Facts: Low corrosion potential Low thermal conductivity and hardness High thermal and mechanical resistances Pashova et al., 2004. J. Agric. Food Chem., 52: 1558-1563. Salazar et al., 2006 J. Agric Food Chem 54: 9955-9958 Packed ZrO 2 column Retentate Permeate Inlet Selectively removes protein (with low flow rates) Can be regenerated (at 500C for 12 h) Proof of concept:

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The Australian Wine Research Institute Lucchetta et al., AJEV, 2013, 64: 400-404 Batch addition of zirconia to model ferments Easy to regenerate (wash with NaOH) Produces heat stable wines No lees production

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The Australian Wine Research Institute Protein Concentration (mg/L) 1 2 3 4 5 6 7 8 Proof of concept Chardonnay wines Carrageenan - Fast Facts: Cell wall hydrocolloid extracted from red seaweeds Used in beer for protein stabilization and copper fining Negatively charged at low pH Cabello-Pasini et al., AJEV 2005; Marangon et al., 2012, AJGWR, 18 (2), 194-202. Removes grape and wine proteins Low sensory and chemical impacts on treated wines

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The Australian Wine Research Institute New polymers are being developed that are specific for haze-forming proteins in wine Stage 1: Know your target www.calendarislandsmainelobster.com Stage 2: Build a custom-designed protein-targeting polymer Criteria: Tolerate wine-like conditions Renewable/recoverable Precipitate with less lees or fit readily into inline systems Approved for food-grade applications X-Ray Crystallography at the Australian Synchrotron to determine the structure of wine proteins Adsorbent resins: New polymers are being developed that are specific for haze-forming proteins in wine

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The Australian Wine Research Institute

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The Australian Wine Research Institute IN WHITE WINES

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The Australian Wine Research Institute Proteases Grape proteins are globular resistant to proteases but proteases CAN work when proteins are in unfolded status

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The Australian Wine Research Institute Heat-unstable grape proteins unfold between 55 and 62 C A suitable protease needs to be active at these elevated temperatures Proteases

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The Australian Wine Research Institute Heat test results ControlBentonite 1.9 g/L ProctaseHeat Heat + Proctase Aspergillopepsin (Proctase) - Fast Facts: Inexpensive food grade proteolytic enzyme Active at acidic pH and high temperatures (50-70 C) Protease 1: Aspergillopepsin Successful pilot trial: Flash pasteurisation + protease Sauvignon Blanc Still need 1.5 g/L Bentonite Only needs 0.2 g/L Bentonite Conditions: Juice flash pasteurisation (1 min @ 70C) with/without 15 mg/L Proctase Marangon et al., Food Chemistry 135 (2012) 11571165

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The Australian Wine Research Institute Selectively removes the majority of haze-forming proteins from wine Cost effective: Significant reduction in the amount of bentonite required Substantially less lees Commercially available Protease 1: Aspergillopepsin X Requires juice to be heated X Requires specialized equipment Currently waiting on approval for use in winemaking...

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The Australian Wine Research Institute Protease 2: BcAp8 min 051015202530 mAU -100 0 100 200 300 400 min 051015202530 mAU -100 0 100 200 300 400 No infection Botrytis Infection Girbau, Stummer, Pocock, Baldock Scott and Waters, AJGWR 2004 Haze-forming proteins HPLC Chromatogram

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The Australian Wine Research Institute Small scale proof of concept: Addition of BcAP8 to juice prior to inoculation. After 3 weeks at 22C, significant decrease in concentration of chitinase Van Sluyter et al., Journal of Agricultural and Food Chemistry, 2013, 61: 9705-9711 Protease 2: BcAp8 Trials are continuing to confirm that this relates to wine stability

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The Australian Wine Research Institute New and improved proteases The search is continuing for enzymes that degrade proteins in winemaking conditions

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The Australian Wine Research Institute The next big thing?

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The Australian Wine Research Institute Summary Recent developments in wine stability: 1. Improve bentonite efficiency Inline dosing Centrifugation and wine recovery from lees 2. Alternative protein adsorbents Zirconia trials continuing to ensure long-term stability Carrageenan trials continuing to ensure long-term stability New protein-targeting polymers to be developed 3. Proteases to degrade haze-forming proteins Aspergillopepsin (Proctase) Requires flash pasteurization, approval for wine addition BcAP8 Works in winemaking conditions trials are continuing for proof of concept Antartic fungi Screening continues for enzymes that are effective in winemaking conditions

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The Australian Wine Research Institute Acknowledgements Matteo Marangon Paul Smith Liz Waters Ken Pocock Simon Schmidt Ella Robinson Vanessa Stockdale Steve Van Sluyter 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.