Lactic Acid Bacteria Spoilage Lucy Joseph Department of
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 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.
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 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) 316 -327
Spoilage Compounds Produced by Lactics Bacteria Compound Sensory Effect Threshold LAB Acetic Acid Vinegar, pungent, sour 0. 2 ppt LAB Ethyl acetate Nail polish remover 7. 5 ppm Lb. , Oeno. Diacetyl Butter, nutty, caramel 0. 1 to 2 ppm Lb. , Pd. 2 -Ethoxy-3, 5 -hexadiene Geranium leaves 0. 1 ppb Lb. , Oeno. 2 -Acetyltetrahydropyridine Mousy 4 to 5 ppb Lb. , Oeno. 2 -Ethyltetrahydropyridine Mousy 2 to 18 ppb Lb. , Oeno. 2 -Acetyl-1 -pyrroline Mousy 7 to 8 ppb Lb. , Pd. Acrolein (+anthocyanin) Bitter Pd. b-D-Glucan Ropy, viscous, oily Oeno. Mannitol Viscous, sweet LAB Skatole (indole) Fecal LAB Biogenic Amines None (headache) Letters in Applied Microbiology 48 (2009) 149– 156 ; E. J. Bartowsky 1. 7 ppm (1. 8)
Metabolic Pathways
Metabolic Pathways (Indole and Skatole) Skatole
Metabolic Pathways (Biogenic Amines)
Preventing Bacterial Spoilage “Best Practices” • Wine is a hostile environment for bacteria – p. H 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. 5 x MRS with 100 ml/liter of V 8 juice) • Lactobacillus and Pediococcus will grow on MRS • Nystatin or cycloheximide will prevent most yeast growth
Q-PCR SYBR Green PCR Chemistry 1. Target Gene 2. PCR 3. SYBR Green binds
1000 cells 100 cells Fluorescence 10000 cells 10 cells threshold 5 10 15 PCR Cycle 20 25
CT-cycle Quantitative PCR Cells per m. L
Preventing Bacterial Spoilage • Cleaning and Sanitation üRemove bacteria and biofilms üKill bacteria
Biofilm Formation u A conditioning film occurs at a liquid interface u Adhesion of cells u Biofilm forms and spreads
Preventing Bacterial Spoilage • Chemical additions üSulfur Dioxide üLysozyme üDMDC (Velcorin) • Physical Treatments üFining üFiltering
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 Lab American Vineyard Foundation California Competitive Grants Volunteers
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