Jonathan R. Cave University of California, Davis Viticulture and Enology

 Yeast Metabolism/Utilization of Oxygen  Oxygen requirements  Lipid synthesis for plasma membrane integrity 1,2  Brewing specification – Strictly monitored/controlled  Yield different aroma and flavor product depending upon available oxygen  Molecular Reactions for Flavor and Aroma  Iron activation to superoxide  Quinone Activation/Fenton reaction  Peroxide radical – highly reactive  Saturation is 8200 ppb 3 Oxygen’s Role in Fermentation

Clark SensorFluorescence Quenching  Optical Sensor  475 nm Fiber Optic (Blue)  600 nm Fluorescence (Orange)  Sanitizable/Autoclavable  High chemical tolerances  Physically Divided or Direct  Non-Invasive  No consumption  Polarographic (800 mV) Electrode  Traditional Dip-Probe  Developed by L. C. Clark (1956)  O 2 Permeable Membrane  Amperometric Ag/AgCl  Movement - Dip, In-Line Flow  Invasive  Consumes O 2 – Negligible Sensors

 Requires Flow or Mixing  O 2 Diffuses through membrane  2 diffusion processes (Membrane and Solution)  Membrane < 20µm so that equilibration across membrane is time limiting rather than the reaction  seconds equilibration  Stable in under 1 minute Clark Sensor Polarographic Electrode

Clark Fluorescence  Temperature Sensitive (External Compensation) Acetone, Toluene, Chloroform, Methylene Chloride, Chlorine Gas, Organic Vapor  Temperature Technically (Internal Thermistor) H 2 (g) SO 2, H 2 S Replenish Electrolyte Interferences

 475 nm Fiber Optic  Excites Fluorescent Dye  FOXY – Hydrophobic Sol-Gel  Pt-porphyrin  Fluorescent Dye in Polymer Matrix  600 nm Fluorescence  Dynamic Fluorescence Quenching Collision of O 2 with fluorophore causes “non-radiative energy transfer” exciting O 2 into triplet state Fluorescence Quenching 5

Experimental Relevance  0.5cm, Physically Divided (Sight Glass)  Flow Rate Independent  pH, CO 2, H 2 S, SO 2, Ionic Species  Chemical Tolerance  NaOH, H 2 O 2, HCl  CIP - autoclave, steam  Linear Range ppb  Accuracy ± 1 ppb  LOD: 1 ppb  Minimal Cross Sensitivity  Yes: Acetone, Chlorine Gas  No: CO 2, H 2 S, SO 2  Compatible with Ethanol PreSens Oxygen Sensor Spots 4 Winery Applicability

Goal Comprehensive model of oxygen availability, necessity, benefit, and detriment from vine to glass Oxygen Management in Winery Operations Jonathan Cave, Nick Gislason, Andrew Waterhouse Cap Manipulation Racking Crush Pressing Barreling Down Bottling

Aerative Pumpovers Splash Racking, Rack and Return, Delestage-ish  High Anticipated Oxygen Solvation  Desired Oxygen Uptake  Early in Fermentation - Low EtOH/High Sugar  SO 2 - Oxygen scavenger and Interaction Inhibitor? Winery Operations

 Observed 29 Pumpovers  23 Aerative  6 Closed Controls  Within first 3 days of fermentation  Pumpovers by experienced cellar staff  Well practiced technique  Not harvest interns  No alteration by experimenters  No interference in the production process  Required Observational Treatments Experimental Design

Oxygen Sensor Spots– Paired Values

 Drop – Distance from Screen to Wine  Splash – Radius and Walls  Flow Rate – From Racking Arm  Flow Type – Screen interaction Parameters Two Conditions  Drop – Large/Small  10” vs. 4”  Splash – Intense/Mild  Spread and Arcing  Flow Rate – Fast/Slow  Flow Type – Turbulent/Laminar

 Range: ppb  Closed PO Control – 0 ppb  Drop – Most Relevant  STDEV of lower [O 2 ] too high  CV > 75% Oxygen Solvation/Assimilation Data Oxygen Assimilation for main observable Treatments SplashFlow RateFlow TypeDrop IntenseMildFastSlowTurbulentLaminarLargeSmall Average (ppb) STDEV t-Test: Two-Sample Unequal Variances LargeSmall Mean Variance Observations 9328 df 119 t Stat 14.3 P(T<=t) one-tail 5.4x t Critical one-tail 1.66 P(T<=t) two-tail 1.1x t Critical two-tail 1.98

Non-Separable Treatments  Coincident Treatments  Interdependence of Rate, Type and Splash  Cannot discern combination of effects or sole influence  Drop is the only separable Parameter  This is not to say they are irrelevant – need more data Data Analysis Treatment Occurrence Turbulent with Large Drop 95% Turbulent with Small Drop 5% Laminar with Large Drop 77% Laminar with Small Drop 23% Total Turbulent 27% Total Laminar 73%

Experimental Variation of Large Drop  We should expect no significant difference  Enough variability that operations are unpredictable  Distinct groups within the single treatment  Combination of effects may attribute to variation  Refinement of current technique is necessary Variability Large Drop Treatment ANOVA DfSum SqMean SqF valuePr(>F) Experiment < 2.2e-16 *** Residuals Experiment Average (ppb) Statistical Group a a ab ab ab ab bc bc bc bc cd cd cde de e

 Variability is immense  Multiple control parameters influence oxygen exposure and solvation  Despite exceptional technique by experienced cellar staff, best control possible, oxygen assimilation into wine by aerative pump over, a commonly employed technique, is inconsistent and capricious.  Technique must be improved  Oxygen levels throughout production are largely uninvestigated  Industry Survey to determine variability  Refinement capability or Operational paradigm shift  Experimentation under strict experimental controls during all winery operations at UC Davis winery  Future collaborations are needed Conclusions and Future Work

Nick Gislason Andrew Waterhouse References 1.) Andreasen, A. A., & Stier, T. J. B Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. Journal of Cellular and Comparative Physiology, 41, 23–36 2.) Andreasen, A. A., & Stier, T. J. B Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. Journal of Cellular and Comparative Physiology, 43, 71–281 3.) Ough, C.S. and M.A. Amerine Methods for analysis of musts and wines, 2nd, Wiley, New York. 4.) Huber, C., T.-A. Nguyen, C. Krause, H. Humele and A. Stangelmayer Oxygen ingress measurement into pet bottles using optical-chemical sensor technology. BrewingScience ) Acknowledgments

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