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