T4 Bacteria Associated with Spoilage in Wine

Question 1

Briefly explain what the fault Acidification (by LAB) is

Answer 1

formation of excessive amounts of acetic acid and D-lactic acid by homo and heterofermentative LAB.

Question 2

Briefly explain how Bitterness spoilage comes about (by LAB)

Answer 2

Few strains of LAB degrade glycerol to acrolein.
Acrolein reacts with phenolics to produce the bitter taint.

Red wines more at risk due to higher phenolic content.
P. parvalus, Leuconostoc mensenteriodes associated.
also called amertume.

Question 3

What compound causes buttery taint and how does it come about?

Answer 3

Diacetyl.

LAB produce diacetyl from pyruvate (from hexose and pentose sugars), but mostly from citric acid (acetic acid also produced

Question 4

My wine smells of Geranium. What has caused this and what is the compound responsible for this disgraceful geranium smell?

Answer 4

LAB have likely metabolised sorbic acid to sorbinol, which reacts with ethanol (plus a few more steps) to form 2-ethoxy-3,5-hexadiene.

This highlights the need for SO2 in sweet wines that we have sorbic acid added. Sorbic acid controls the growth of yeast.

Question 5

Why is the production of biogenic amines a concern?
How do they form?
What can be done to reduce their levels in wine?

Answer 5

The most common one is histamine- causes allergic reactions and hypertension.

Formed by LAB that have amino acid decarboxylases. can be present at 3.4 and 1.1 mg/L in red and white wines. Higher in wines having undergone MLF.

Can be reduced with the selection of LAB strains that lack the ability to produce amines, and limit the growth of wild LAB.

Question 6

Mannitol taint- What is it and how is it formed?

Answer 6

Sugar alcohol. Sensory effect- viscous, ester-like combined with a sweet and irritating finish.

The Heterofermentative bacteria L. brevis produces mannitol from the reduction of fructose and fructose-6-phosphate.

Rare, although more common in dessert wines with high pH.

Question 7

What is the source of a Mousy off-flavour in wine?

Why is it there only a delayed detection after swallowing

Answer 7

Formed by a variety of LAB. Several compounds including acetyltetrahydropyridine and 2-acetyl-1-pyrroline. PPB detection threshold. Possible formed from lysine or ornithine.
Often attributed to Brettanomyces but also from the LAB Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus hilgardii

Delayed detection due to alkaline conditions in the mouth which increases the volatility of the compounds involved.

Question 8

What is ropiness?

How does this taint come about?

Answer 8

Gives the wine an oily, viscous mouthfeel- slimy and fatty.
Due to the production of dextrins and other polysaccharides by some LAB spp. Thes include Leuconostoc mesenteroides, Pediococcus damnosus.

Question 9

How does the degradation of tartaric acid come about? Is this by homo or heterofermentative LAB strains?

Answer 9

Both. Homofermentative L. plantarum breaks tartaric down into lactic and acetic acid.
Heterofermentative Lactobacillus brevis breaks it down into acetic acid and succinic acid.

Rare fault, more common at pH>3.65.

Question 10

Under what conditions is acidification spoilage most likely to occur?
What substrate is utilised?

Answer 10

• Where there is residual sugar after primary fermentation (as this is done by LAB).
• hexose and pentose sugars. Can be via the metabolism of citric acid.

Question 11

Why are red wines more at risk of bitterness taint?

Answer 11

They have higher a higher phenolic content.
Glycerol is broken down to propionaldehyde which is dehydrated to Acrolein.
Acrolein is tasteless but reacts with phenolics to produce the bitter taint.

Question 12

What is the difference between the 2 pathways by which diacetyl is produced?

Answer 12

The origin of pyruvate.
One source of pyruvate is from the metabolism of pentose and hexose sugars, and the other source is from the metabolism of citric acid. This later pathway produces acetic acid as citric acid is broken down into oxaloacetate and acetic acid. Oxaloacetate is then broken down into pyruvic acid.

Citric acid metabolism is the most likely mechanism of diacetyl production.

Question 13

What is a common method for reducing diacetyl concentration in a wine?
Why does it work?
What else can this work for?

Answer 13

Adding a small amount of grape juice and some wine yeast.
This can work because yeast can reduce diacetyl to acetoin and butandiol, both of which have higher detection thresholds.
Also works for acetaldehyde as when the yeast has exhausted all fermentable sugars they can break down acetaldehyde for ATP.

Question 14

What are the factors that affect diacetyl production?

Answer 14

• Strain (Oenocuccus produces least, Lacto and Pedoi more)
• temperature
• pH (low pH favours higher, as leads to a slower growth rate).
• oxygen tension
• presence of viable yeasts.
• Citric acid concentration (more=higher diacetyl)
• LAB inoculation rate.
• Timing of inoculation.

Question 15

What effect on diacetyl production does biomass accumulation of LAB have?
What factors affect this?

Answer 15

Biomass accumulation is linked to diacetyl production through citric acid degradation. Slower growth rates also lead to higher diacetyl.

• Low inoculation rates necessitate biomass production.
• low pH leads to slower growth of O. oeni and this leads to higher diacetyl.

Question 16

Why is the timing of LAB inoculation a factor in diacetyl production?

Answer 16

Yeast can utilise diacetyl during fermentation and metabolise it.
If diacetyl is formed during the reductive fermentation conditions or on lees then it can be reduced to acetoin and butandiol.
Production in some strains is only possible in presence of fermentable sugars, other strains citrate uptake

Question 17

Explain the taxonomy and characteristics of acetic acid bacteria.
How might you easily distinguish a culture of these bacteria from LAB?

Answer 17

• Family Acetobacteraceae
• Gram-negative rods.
• Strict Anaerobes
• 2 genera Gluconobacter and Acetobacter relevant to wine.
• Can oxidise ethanol to acetic acid.

Distinguish from LAB using the humble gram stain.

Question 18

What media is useful for the isolation of acetic acid bacteria?

Answer 18

Glucose yeast extract Carbonate (GYC) Agar. Clearing zone suggests AAB.

Question 19

How does the proportion of Acetobacter spp. and Gluconobacter oxydans change from undamaged to damaged berries?

Answer 19

Undamaged berries- G.oxydans predominates 1E2/g
Slight damage- Acetobacter start to be able to be isolated.
damaged fruit- Acetobacter spp predominate (45-85%) over G. oxydans. 1E5 -1E6 cells of AAB/ml.

Question 20

What factors influence the growth of AAB in primary fermentation?

Answer 20

• Speed of fermentation- anaerobic nature of fermentation will see a large drop in viable AAB numbers. therefore fast red ferments will see a greater drop.
• Amount of damage in fruit. damaged fruit will go into primary ferment with higher numbers of AAB.
• Ratio of AAB to S. cerevisiae. less VA results from a higher relative number of S. cerevisiae.

SO- Acetobacter replace G. oxydans during vinification and are the only species present at the end of alcoholic fermentation.

Question 21

When is the growth of AAB a concern? what stimulates this growth?

Answer 21

During conservation.

• Oxygen ingress from pumping and barrel ageing stimulate this growth, especially at wine/air interface. can reach 1E4 to 1E5 cells/ml.
• Ethanol concentration- fewer strains are able to tolerate high ethanol conc. strain variation.
• Relatively tolerant to so2.
• pH affects the level of ethanol that can be tolerated. As pH decreases, so does ethanol tolerance.

Question 22

How does acetic acid form in wine?

Answer 22

Ethanol–>acetaldehyde–> Acetic acid.

2 enzymes- ethanol dehydrogenase and acetaldehyde dehydrogenase.

Question 23

How does acetaldehyde accumulate in wine if it is an intermediate in the acetic acid pathway?

Answer 23

Ethanol–>acetaldehyde–> Acetic acid.
2 enzymes- ethanol dehydrogenase and acetaldehyde dehydrogenase.

• High ethanol decreases the stability of acetaldehyde dehydrogenase. leading to an accumulation of acetaldehyde.
• Free SO2 binds acetaldehyde, encouraging its formation.
• Low O2 conditions favour the formation of acetaldehyde.

Question 24

How does dihydroxyacetone form in wine and what is the effect on wine?

Answer 24

-G. oxydans and A. aceti oxidise glycerol to dihydroxyacetone. Only occurs in grapes but can persist through to the final wine.

Reaction with certain amino acids can yield a crust like aroma. Also reduces glycerol in wine and therefore those positive sensorial characteristics are lost.
Can bind FSO2 and reduce the antimicrobial effect.

Question 25

What is the pathway that AAB metabolise hexose and pentose sugars?
What are the products and how does pH affect this?

Answer 25

Hexose monophosphate pathway.

in G. oxydans,

• below pH 3.5, gluconic acid the main product.
• above pH 3.5 gluconic acid oxidised to ketogluconic acid.

Metabolism of sugars by A. aceti and A. pasteuranus is poor, but the products are the same as above.

Question 26

What organic acids can AAB metabolise? what is the end product?

Answer 26

Acetobacter can consume lactic, fumaric, succinic, citric, and malic acids.
G. oxydans can only consume pyruvic and lactic acids (missing TCA cycle enzymes).

Variety of end products including acetic acid and acetoin (buttery character).

Question 27

What is the best way to control AAB in wine production>

Answer 27

eliminate oxygen as much as possible, such as from ullages etc.
proper sanitation of all equipment coming into contact with wine.

Question 28

What are the spore-forming bacteria that can lead to wine spoilage?

Answer 28

Bacillus and Clostridium.

Bacillus- spoilage is rare and has occurred in dessert wines. Increase in VA.
Clostridium- butyric acid taint (also present in parmesan cheese). More likely in high pH wines. Rare.

Question 29

Actinomyces and streptomyces have been implicated in what taint?

Answer 29

Cork taint- musty aroma.