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

2. Review of Human Olfactory Sensory Detection

3. 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

4. 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

5. Difference in Panelists Perception of Standards

6. Difference in Panelists Perception of the Same Sample

7. Difference in Odor Perception of One Panel Member on Different Days

8. 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

9. 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

10. For Example….

11. 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

12. 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) 316-327

13. 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

14. Metabolic Pathways

15. Metabolic Pathways (Indole and Skatole) Skatole

16. Metabolic Pathways (Biogenic Amines)

17. 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

18. Writing about spoiled wines by lactic acid bacteria:

19. Monitoring Lactic Acid Bacteria Microscopic examination Plating Q-PCR

20. Images of Lactic Acid Bacteria Pediococcus  Oenococcus   Lactobacillus

21. 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

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

23. PCR Cycle Fluorescence 515102025 10000 cells 1000 cells 100 cells 10 cells threshold

24. Cells per mL CT-cycle Quantitative PCR

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

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

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

28. 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)

29. 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

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