Lactic Acid Bacteria Spoilage Lucy Joseph Department of Viticulture and Enology U.C. Davis

Review of Human Olfactory Sensory Detection

How Do We Perceive Aroma? Every olfactory receptor binds many similar odorants with different affinities. Olfactory sensory neurons are directly connected to the olfactory bulb which is connected directly to the primitive brain or the limbic system. This system is involved in processing memory and emotion. 10 million olfactory sensory neurons on the olfactory epithelium

Genetics of Olfaction Humans have 388 genes that code for olfactory receptors (OR) and about 414 pseudogenes These genes have different alleles but only one allele is expressed at a time OR genes are divided into 17 families and 127 subfamilies based on sequence and protein structure Each receptor reacts with one type of chemical or chemical constituent “Aroma” often consists of a mixture of these signals to receptors that we learn to associate with a given object like bacon or coffee

Difference in Panelists Perception of Standards

Difference in Panelists Perception of the Same Sample

Difference in Odor Perception of One Panel Member on Different Days

Lactic Acid Bacteria Found in Wine Lactobacillus – Lb. brevis, Lb. casei, Lb. hilgardii, Lb. plantarum, Lb. lindneri, Lb. kunkeei Pediococcus – Pd. damnosus, Pd. parvulus, Pd. ethanolidurans Oenococcus – O. oeni

Where do they come from? Populations may become established in your winery and can be hard to dislodge Vineyards may be reservoirs for some species

For Example….

Bacteria Found on Grapes Australia - MLAB Enrichment with Plating and PCR Lactobacillus - Cabernet Sauvignon, Merlot, Semillon, Sauvignon Blanc Lactococcus - Sauvignon Blanc Enterococcus - Merlot, Pinot Noir, Semillon, Sauvignon Blanc Weissella – Semillon S. Bae, G.H. Fleet and G.M. Heard. Journal of Applied Microbiology 100 (2006) 712–727

Bacteria Found on Grapes France - Plating and PCR Oenococcus Gluconobacter Pediococcus Renouf, Vincent, Olivier Claisse, Aline Lonvaud-Funel, Australian Journal of Grape and Wine Research, 11 (3)

Spoilage Compounds Produced by Lactics BacteriaCompoundSensory EffectThreshold LABAcetic AcidVinegar, pungent, sour0.2 ppt LABEthyl acetateNail polish remover7.5 ppm Lb., Oeno.DiacetylButter, nutty, caramel0.1 to 2 ppm Lb., Pd.2-Ethoxy-3,5-hexadieneGeranium leaves0.1 ppb Lb., Oeno.2-Acetyl- tetrahydropyridine Mousy4 to 5 ppb Lb., Oeno.2-EthyltetrahydropyridineMousy2 to 18 ppb Lb., Oeno.2-Acetyl-1-pyrrolineMousy7 to 8 ppb Lb., Pd.Acrolein (+anthocyanin)Bitter Pd.b-D-GlucanRopy, viscous, oily Oeno.MannitolViscous, sweet LABSkatole (indole)Fecal1.7 ppm (1.8) LABBiogenic AminesNone (headache) Letters in Applied Microbiology 48 (2009) 149–156 ; E.J. Bartowsky

Metabolic Pathways

Metabolic Pathways (Indole and Skatole) Skatole

Metabolic Pathways (Biogenic Amines)

Preventing Bacterial Spoilage “Best Practices” Wine is a hostile environment for bacteria – pH 3.6 or less – Ethanol up to 16% – High levels of phenolic compounds – SO 2 addition at crush Cold Storage at 15 o C (60 o F) Avoid Stuck Fermentations Carefully control nutrient additions

Writing about spoiled wines by lactic acid bacteria:

Monitoring Lactic Acid Bacteria Microscopic examination Plating Q-PCR

Images of Lactic Acid Bacteria Pediococcus  Oenococcus   Lactobacillus

Plating on Selective Media We use MLAB (0.5x MRS with 100 ml/liter of V8 juice) Lactobacillus and Pediococcus will grow on MRS Nystatin or cycloheximide will prevent most yeast growth

1. Target Gene 2. PCR 3. SYBR Green binds Q-PCR SYBR Green PCR Chemistry

PCR Cycle Fluorescence cells 1000 cells 100 cells 10 cells threshold

Cells per mL CT-cycle Quantitative PCR

Preventing Bacterial Spoilage Cleaning and Sanitation Remove bacteria and biofilms Kill bacteria

Biofilm Formation  A conditioning film occurs at a liquid interface  Adhesion of cells  Biofilm forms and spreads

Preventing Bacterial Spoilage Chemical additions Sulfur Dioxide Lysozyme DMDC (Velcorin) Physical Treatments Fining Filtering

New Techniques on the Horizon Bacteriocins (with sulfur dioxide) High pressure processing Ultrasound at high power Flash heating Pulsed electrical fields UV Irradiation (white wine)

Summary Lactic acid bacteria are often a problem when winemaking conditions are not ideal Traditional practices are designed to keep bacterial spoilage under control Deviation from “Best Practices” can produce unexpected results and spoilage Cleaning and sanitation practices are crucial to controlling bacterial contamination

Acknowledgments Linda Bisson Bisson Lab American Vineyard Foundation California Competitive Grants Volunteers