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 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) 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
– 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
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 mL
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