Recent development in removing protein haze from white wines

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

2. Wine Haze

3. Wine Haze is caused by… Protein aggregation... With sulfate + Polysaccharides + Phenolics + Organic acids ...and more...!

4. Haze-forming proteins Grape proteins that are resistant to winemaking Grape Invertase Glucanase Chitinases Thaumatin-Like Proteins Grape Pathogenesis-Related (PR) proteins Thaumatin-Like Proteins Lipid Transfer Proteins

5. Haze-forming proteins Grape Pathogenis-Related (PR) proteins increase in concentration with infection 0.40 0.35 0.30 0.25 Wine haze after heat test (Abs 540) 0.20 0.15 0.10 0.05 control 1-5 10-30 >30 Percent of powdery mildew infection Girbau, Stummer, Pocock, Baldock Scott and Waters, AJGWR 2004

6. Bentonite… CRAZY

7. Bentonite: the good The good • Cheap • Readily available • Easy to use • Non-toxic Effective mechanism:

8. Bentonite: the bad • Non-selective • Extraprocessing steps: • water addition • settling time • waste removal • Badpress...? 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

9. Bentonite: the bad $1 Billion wasted worldwide Majewski, P.; Barbalet, A.; Waters, E. J. The Australian & New Zealand Grapegrower & Winemaker2011, 58–62.

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

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

12. Bentonite: the crazy alternatives • Criteria for bentonite alternatives: • Better settling – less waste • More efficient – use less material • More selective • Cost effective • No flavour taint

13. Bentonite: the crazy alternatives Adsorbent resins Carrageenan Chitin Phytic acid Zirconia Bromelain

14. Retentate Inlet Packed ZrO2 column Permeate • Zirconia - Fast Facts: • Low corrosion potential • Low thermal conductivity and hardness • High thermal and mechanical resistances Proof of concept: • Selectively removes protein (with low flow rates) • Can be regenerated (at 500ºC for 12 h) • Pashova et al., 2004. J. Agric. Food Chem., 52: 1558-1563. Salazar et al., 2006 J. Agric Food Chem 54: 9955-9958

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

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

17. New polymers are being developed that are specific for haze-forming proteins in wine • Adsorbent resins: • New polymers are being developed that are specific for haze-forming proteins in wine Stage 1: Know your target X-Ray Crystallography at the Australian Synchrotron to determine the structure of wine proteins • 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 www.calendarislandsmainelobster.com

19. PROTEASES IN WHITE WINES

20. Proteases • Grape proteins are globular  resistant to proteases • …but • proteases CAN work when proteins are in unfolded status

21. Proteases Heat-unstable grape proteins unfold between 55 and 62 °C A suitable protease needs to be active at these elevated temperatures

22. Protease 1: Aspergillopepsin • Aspergillopepsin (Proctase) - Fast Facts: • Inexpensive food grade proteolytic enzyme • Active at acidic pH and high temperatures (50-70 °C) Successful pilot trial: Flash pasteurisation + protease Conditions: Juice flash pasteurisation (1 min @ 70°C) with/without 15 mg/L Proctase Heat test results Heat + Proctase Control Bentonite 1.9 g/L Proctase Heat Still need 1.5 g/L Bentonite Only needs 0.2 g/L Bentonite Sauvignon Blanc Marangon et al., Food Chemistry 135 (2012) 1157–1165

23. Protease 1: Aspergillopepsin • 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 • Requires juice to be heated • Requiresspecialized equipment • Currently waiting on approval for use in winemaking...

24. mAU 400 300 200 100 0 -100 0 5 10 15 20 25 30 min mAU -100 0 5 10 15 20 25 30 min Protease 2: BcAp8 Haze-forming proteins HPLC Chromatogram No infection 400 Botrytis Infection 300 200 100 0 Girbau, Stummer, Pocock, Baldock Scott and Waters, AJGWR 2004

25. Protease 2: BcAp8 • Small scale proof of concept: • Addition of BcAP8 to juice prior to inoculation. • After3 weeks at 22°C, significant decrease in concentration of chitinase • Trials are continuing to confirm that this relates to wine stability Van Sluyter et al., Journal of Agricultural and Food Chemistry, 2013, 61: 9705-9711

26. New and improved proteases The search is continuing for enzymes that degrade proteins in winemaking conditions

27. The next big thing? FUNGI

28. 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

29. 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.