Post on 03-Jan-2016
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Lactic Acid Bacteria Spoilage
Lucy JosephDepartment 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 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
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
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-Acetyl-tetrahydropyridine
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 1.7 ppm (1.8)
LAB Biogenic Amines None (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– SO2 addition at crush
• Cold Storage at 15o C (60o 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
Flu
ore
scen
ce
5 1510 20 25
10000 cells
1000 cells
100 cells
10cells
threshold
Cells per mLCells per mL
CT
-cyc
leC
T-c
ycle
Quantitative PCR
Preventing Bacterial Spoilage• Cleaning and Sanitation
Remove bacteria and biofilmsKill bacteria
Biofilm Formation
A conditioning film occurs at a liquid interface
Adhesion of cells
Biofilm forms and spreads
Preventing Bacterial Spoilage
• Chemical additionsSulfur DioxideLysozymeDMDC (Velcorin)
• Physical TreatmentsFiningFiltering
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