kenneth, eriko, zach, jihee
DESCRIPTION
Inactivation of Wine Spoilage Yeasts Dekkera bruxellensis Using Low Electric Current Treatment(LEC ). Kenneth, Eriko, Zach, Jihee. Abstract. Purpose: Inactivate yeast Dekkera bruxellensis by low electric current treatment (LEC) - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/1.jpg)
Inactivation of Wine Spoilage Yeasts Dekkera bruxellensis Using Low Electric Current Treatment(LEC)
Kenneth, Eriko, Zach, Jihee
![Page 2: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/2.jpg)
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
![Page 3: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/3.jpg)
Abstract (cont’d)
• Conclusion: Dekkera bruxellensis was inhibited by LEC.
• Significance of the study: LEC can potentially be used in the wine industry
![Page 4: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/4.jpg)
Introduction• Winemaking–Result of multiplication and
metabolism of yeasts and bacteria in grape juice.–MO elimination after
fermentation
![Page 5: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/5.jpg)
Intro: Wine Spoilage • During aging or after bottling • Unpleasant odors and taste by Dekkera –Volatile phenols–Acetic acid–Tetrahydropyridines
![Page 6: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/6.jpg)
Intro: Current treatment
• Sulfur dioxide (SO2)
–Antioxidant–Antimicrobial–Negatively affect human health
![Page 7: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/7.jpg)
Intro: Focus of Experiment
• Yeast Dekkera bruxellensis 4481 strain • Low electric current(LEC) technology
Goal: - Reduction of SO2
- Elimination of SO2
![Page 8: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/8.jpg)
Materials and Methods1. Strain Maintenance, Growth, and Monitoring Culturability and Viability.
2. Equipment and Apparatus for LEC Treatment3. Experimental Procedure
4. Chemical and Physical Analysis and Statistics
![Page 9: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/9.jpg)
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
![Page 10: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/10.jpg)
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
![Page 11: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/11.jpg)
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
![Page 12: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/12.jpg)
2. Equipment and Apparatus for LEC Treatment
2L of wine+/- +/-
200 mA
19cm
20cm
15cm
![Page 13: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/13.jpg)
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
![Page 14: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/14.jpg)
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
![Page 15: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/15.jpg)
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)
![Page 16: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/16.jpg)
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
![Page 17: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/17.jpg)
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)
![Page 18: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/18.jpg)
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)
![Page 19: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/19.jpg)
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
![Page 20: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/20.jpg)
![Page 21: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/21.jpg)
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
![Page 22: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/22.jpg)
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
![Page 23: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/23.jpg)
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
![Page 24: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/24.jpg)
Figure 8. Scanning electron micrograph of Dekkera bruxellensis 4481 strain
![Page 25: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/25.jpg)
Part 1 : Experiment Without Adaptation
Grown in absence of ethanol
![Page 26: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/26.jpg)
Figure 1. Survival dynamics of Dekkera bruxellensis cells in red wine
![Page 27: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/27.jpg)
Figure 2. Mean ATP content in red wine
![Page 28: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/28.jpg)
Volatile phenols in wine after 60 days Concentration (mg L-1)
Test p-Coumaric acidFerulic
acid4-Ethyl-phenol
4-Ethyl-guaiacol
i 0.35 0.12 0.18 0.15
ii 0.57 0.18 0.16 0.11
iii 1.90 0.40 0.05 0.05
iv 1.85 0.35 0.05 0.05
v 1.89 0.42 0.05 0.05
![Page 29: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/29.jpg)
Part 2 : Experiment With Adaptation
Cells grown on 10% ethanol
![Page 30: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/30.jpg)
Figure 4. Survival dynamics of Dekkera bruxellensis cells
![Page 31: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/31.jpg)
Figure 5. Mean ATP content in red wine
![Page 32: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/32.jpg)
Figure 6. Kinetics of volatile phenol accumulation
![Page 33: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/33.jpg)
Kinetics of biogenic amine accumulation
![Page 34: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/34.jpg)
i ii iii iv units Total
acidity 6.2 5.4 5.4 5.6 g L-1
Volatile acidity 1.2 0.1 0.2 0.6 g L-1
Oenological parameters of wine after 60 days.
![Page 35: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/35.jpg)
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
![Page 36: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/36.jpg)
Discussion• Similar Effects from SO2 vs. LEC (Dekkera bruxellensis strain 4481)–Viability –Limits wine deterioration•Morphology change(Irreversible)• Volatile acid • APT content
![Page 37: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/37.jpg)
Discussion• Additional– ATP content in EWOA phenomenon • Cross-contamination
– To improve experiment in future • Extend stain and fermentation
![Page 38: Kenneth, Eriko, Zach, Jihee](https://reader035.vdocuments.mx/reader035/viewer/2022062305/56816415550346895dd5c817/html5/thumbnails/38.jpg)
Conclusion• Keep good quality –Replace ‘chemical additives’ to ‘Organic’ wine
• LEC is GOOD for inactivating wine spoilage yeast D. bruxellensis (CBS 4481)