1. Inactivation of Wine Spoilage Yeasts Dekkera bruxellensis Using Low Electric Current Treatment(LEC ) Kenneth, Eriko, Zach, Jihee

2. Abstract Purpose: Inactivate yeast Dekkera bruxellensis by low electric current treatment (LEC) Methods and Results: Applied LEC for 60 days, and limited the growth of the yeast and deterioration of wine

3. Abstract (cont’d) Conclusion: Dekkera bruxellensis was inhibited by LEC. Significance of the study: LEC can potentially be used in the wine industry

4. Introduction Winemaking – Result of multiplication and metabolism of yeasts and bacteria in grape juice. – MO elimination after fermentation

5. Intro: Wine Spoilage During aging or after bottling Unpleasant odors and taste by Dekkera – Volatile phenols – Acetic acid – Tetrahydropyridines

6. Intro: Current treatment Sulfur dioxide (SO 2 ) – Antioxidant – Antimicrobial – Negatively affect human health

7. Intro: Focus of Experiment Yeast Dekkera bruxellensis 4481 strain Low electric current(LEC) technology Goal: - Reduction of SO 2 - Elimination of SO 2

8. Materials and Methods 1. Strain Maintenance, Growth, and Monitoring Culturability and Viability. 2. Equipment and Apparatus for LEC Treatment 3. Experimental Procedure 4. Chemical and Physical Analysis and Statistics

9. 1. Strain Maintenance, Growth, and Monitoring Culturability and Viability. Pure strain of Dekkera bruxellensis strain 4481 Strain was maintained on solid YPD media at -80 degrees Celcius

10. 1. Strain Maintenance, Growth, and Monitoring Culturability and Viability. To monitor culturability the strain was inoculated on YPD agar in petri dishes To monitor viability a portable bioluminometer biocounter was used. Biocounter was used on Microbial Biomass Test Kits and Standard ATP Assays

11. 2. Equipment and Apparatus for LEC Treatment LEC (low electric current) module was purchased from De Ponti Application Electronics. Metal mixed oxide (MMO) electrodes were used Tests performed in cylindrical polyethylene tanks for 60 days

12. 2. Equipment and Apparatus for LEC Treatment 2L of wine +/- 200 mA 19cm 20cm 15cm

13. 2. Equipment and Apparatus for LEC Treatment 200 mA applied, with the polarity inverted every 60 seconds. Amps and volts monitored by probes and data logger immersed in wine

14. 3. Experimental Procedure From pure stock cultures, 2 batches liquid YPD media were inoculated One batch has 10% ethanol (ABV) and the other has no ethanol Batch 1: Experiment without Adaptation (no ethanol) referred to as EWOA Batch 2: Experiment with Adaptation (with ethanol) referred to as EWA

15. 3. Experimental Procedure After 3-5 days the optical density was measured at 640 nm Cells harvested using centrifugation Washed once with 10g/L peptone Diluted to create stock cultures of 10^6 cfu/ml (confirmed using plate counts)

16. 3. Experimental Procedure Acquired 50L of Montepulcian d’Abruzzo red wine Filtered with.45 um membranes. Wine has ABV of 13.5% at this point. Split into two 25L batches

17. 3. Experimental Procedure The EWOA batch was used to make 5 flasks for testing: 1: wine and cells (control) 2: wine and cells + 80mg/L SO2 3: wine and cells +200 mA of LEC 4: wine and cells +200 mA and 30mh/L SO2 5: wine only (witness)

18. 3. Experimental Procedure The EWA batch was used to make 5 flasks for testing: 1: wine and cells (control) 2: wine and cells + 80mg/L SO2 3: wine and cells +200 mA of LEC 4: wine only (witness)

19. 3. Experimental Procedure Flasks placed in sterile cabinet at 18-20 degrees celcius for 60 days 70 ml of liquid paraffin was added to each flask to prevent oxygen exposure and evaporation All flasks were monitored for pH and temperature changes periodically No stirring

21. 4. Chemical and Physical Analysis and Statistics pH, titratable acidity, ethanol, sugar concentrations were monitored Sensorial characteristics were tested by a panel before and after LEC treatment Panel consisted of University staff with panel experience

22. 4. Chemical and Physical Analysis and Statistics A scanning electron microscope was used to observe cell morphology changes from LEC High Pressure Liquid Chromatography (HPLC) was used to measure volatile phenols and biogenic amines

23. 4. Chemical and Physical Analysis and Statistics All data was expressed by means of tree replications and standard deviations Analyzed with ANOVA Costat-statistics Software v 6.3

24. Figure 8. Scanning electron micrograph of Dekkera bruxellensis 4481 strain

25. Part 1 : Experiment Without Adaptation Grown in absence of ethanol

26. Figure 1. Survival dynamics of Dekkera bruxellensis cells in red wine

27. Figure 2. Mean ATP content in red wine

28. Volatile phenols in wine after 60 days

29. Part 2 : Experiment With Adaptation Cells grown on 10% ethanol

30. Figure 4. Survival dynamics of Dekkera bruxellensis cells

31. Figure 5. Mean ATP content in red wine

32. Figure 6. Kinetics of volatile phenol accumulation

33. Kinetics of biogenic amine accumulation

34. Oenological parameters of wine after 60 days.

35. Discussion Major goal in wine industry -Reducing the risk of wine being spoiled by microbial activity Must undertake a critical analysis of many factors associated with the actual development of spoilage YPD, Microbial Biomass Test Kit, Standard ATP assays

36. Discussion Similar Effects from SO 2 vs. LEC (Dekkera bruxellensis strain 4481) – Viability – Limits wine deterioration Morphology change (Irreversible ) Volatile acid APT content

37. Discussion Additional – ATP content in EWOA phenomenon Cross-contamination – To improve experiment in future Extend stain and fermentation

38. Conclusion Keep good quality – Replace ‘chemical additives’ to ‘Organic’ wine LEC is GOOD for inactivating wine spoilage yeast D. bruxellensis (CBS 4481)