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Lactic Acid Bacteria Spoilage

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?. 10 million olfactory sensory neurons on the olfactory epithelium. Every olfactory receptor binds many similar odorants

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Lactic Acid Bacteria Spoilage

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  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? 10 million olfactory sensory neurons on the olfactory epithelium 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.

  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 GrapesAustralia - 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 GrapesFrance - 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 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 • SO2 addition at crush • Cold Storage at 15o C (60o 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. Q-PCR SYBR Green PCR Chemistry 1. Target Gene 2. PCR 3. SYBR Green binds

  23. threshold 5 10 15 20 25 10000 cells 1000 cells 100 cells 10 cells Fluorescence PCR Cycle

  24. Quantitative PCR CT-cycle Cells per mL

  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

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