Assessments of Microbial Activity in Wine Linda F. Bisson Department of Viticulture and Enology, UCD December 13, 2013

Post-Fermentation Microbial Activity Turbidity CO 2 Production Film Production Polysaccharide Production Positive Character Production Negative Character Production – Odor – Taste – Color – Sediment – Haze

Mechanisms to Assess Post- Fermentation Microbial Presence Microscopically Appearance of Wine – Turbidity – Effervescence – Films Analysis of Metabolic Activity

The Types of Impacts of Microbial Metabolites Positive character synthesis Negative character synthesis Modify existing character Enhance or amplify perception of existing characters Diminish or buffer perception of existing characters Matrix effects: change in chemical properties

Positive Contributions to Aroma Direct synthesis Release of bound molecules Autolysis factors Indirect (chemical) effects Matrix effects

Types of Microbial Transformations of Flavor Compounds Primary roles – Production of flavor compounds de novo from nutrients – Liberation of grape flavor components from precursors Secondary roles – Provide chemical reactants – Enzymatic modification of grape/oak flavors – Impact Redox status and buffering capacity

Microbial Components Impacting Wine Flavor Metabolites Enzymes Catalysts Mannoproteins and Polysaccharides

Direct Synthesis Esters Alcohols Aldehydes S-volatiles Acids

Release of Bound Molecules Thiols in Sauvignon blanc – Cysteine conjugates – Carbon sulfur lyase activity Terpenes – Β-Glucosidase activity Norisoprenoids – Β-Glucosidase activity

Autolysis Factors Release of active enzymes: hydrolases Release of cellular macromolecular components Breakdown of cellular macromolecular components

Indirect (Chemical) Effects Creation of reactive molecules – S-containing compounds – Diacetyl – Aldehydes Alteration of pH Alteration of Redox status of juice

Matrix Effects Alteration of solution chemistry Production of flavor enhancers – Dimethyl sulfide Production of masking factors – Fusel family – Hydrogen sulfide

Post-Fermentation Microbiota ML Fermentation Surface films Brettanomyces spoilage Yeast spoilage

Monitoring Lactic Acid Bacterial Activity in Wine Measure decrease in malate levels Assess change in pH Note presence of effervescence Follow increase in wine turbidity Evaluate aroma profile

Monitoring Lactic Acid Bacterial Activity in Wine Measure decrease in malate levels – Will tell you when it is done Assess change in pH – Something is happening, will not tell you it is complete Note presence of effervescence – Something is producing CO 2 Follow increase in suspended wine turbidity – Something is growing in the wine Evaluate aroma profile – Characteristic compounds of lactic acid bacteria Diacetyl Acetic acid Off-characters

ML Fermentation Monitor disappearance of malate – Paper chromatography – Enzymatic By hand Automated – Spectroscopy UV/VIS FTIR (Fourier Transform Infrared) – Photometric analysis

Note on Measurements Some methods are direct – Interference is minimal – Misidentification of compound rare Some methods are indirect – Need to make sure peak is correctly being identified – Interference can be a problem – Misidentification can occur if unexpected components are present

Lactic Acid Bacteria Off-Odors Mousiness “Poo” off- notes Animal characteristics: fur, meaty Floral taints: geranium Metallic Vomit Rancidity

Mousiness Several compounds(oxidation products of lysine) have been implicated in this off- character: 2,4,6-trimethyl-1,3,5-triazine 2-ethyl-3,4,5,6-tetrahydropyridine 2-acetyl-3,4,5,6-tetrahydropyridine

Lactic Acid Bacteria Off-Tastes Enhanced sourness Bitterness

Monitoring Lactic Acid Bacterial Activity in Wine: Best Practices Microscopic analysis Aroma analysis Taste analysis Monitor malate levels

Surface Films Bacteria or Yeast Most Often Both Off-Character Production Film Microbiota Can Produce Copious Amounts of Off-Characters Due to Availability of Oxygen

Brettanomyces Problem in barrels Can monitor 4-ethyl phenol or 4 ethyl guaiacol formation by nose or analytically Some wines suppress odors from these compounds; the mechanism is not understood Monitor via microscopy; sometimes Saccharomyces looks like Brettanomyces Genomic/molecular monitoring of presence

Spoilage Yeasts Use residual sugar Turbidity and effervescence in bottle Zygosaccharomyces Saccharomyces

Advice Need to know you have the potential for a problem before the off-character appears Some of these off-characters are transient Some characters difficult to remove – Blending out – Charcoal fining Need to be careful to NOT spread it around the winery