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)

• Methods and Results: Applied LEC for 60 days, and limited the growth of the yeast and deterioration of wine

Abstract (cont’d)

• Conclusion: Dekkera bruxellensis was inhibited by LEC.

• Significance of the study: LEC can potentially be used in the wine industry

Introduction• Winemaking–Result of multiplication and

metabolism of yeasts and bacteria in grape juice.–MO elimination after

fermentation

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

Intro: Current treatment

• Sulfur dioxide (SO2)

–Antioxidant–Antimicrobial–Negatively affect human health

Intro: Focus of Experiment

• Yeast Dekkera bruxellensis 4481 strain • Low electric current(LEC) technology

Goal: - Reduction of SO2

- Elimination of SO2

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

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

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

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

2. Equipment and Apparatus for LEC Treatment

2L of wine+/- +/-

200 mA

19cm

20cm

15cm

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

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

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)

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

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)

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)

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

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

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

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

Figure 8.  Scanning electron micrograph of Dekkera bruxellensis 4481 strain

Part 1 : Experiment Without Adaptation

Grown in absence of ethanol

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

Figure 2.  Mean ATP content in red wine

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

Part 2 : Experiment With Adaptation

Cells grown on 10% ethanol

Figure 4.  Survival dynamics of Dekkera bruxellensis  cells

Figure 5.  Mean ATP content in red wine

Figure 6.  Kinetics of volatile phenol accumulation

Kinetics of biogenic amine accumulation

  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.

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

Discussion• Similar Effects from SO2 vs. LEC (Dekkera bruxellensis strain 4481)–Viability –Limits wine deterioration•Morphology change(Irreversible)• Volatile acid • APT content

Discussion• Additional– ATP content in EWOA phenomenon • Cross-contamination

– To improve experiment in future • Extend stain and fermentation

Conclusion• Keep good quality –Replace ‘chemical additives’ to ‘Organic’ wine

• LEC is GOOD for inactivating wine spoilage yeast D. bruxellensis (CBS 4481)