Topic 3- Impact of yeasts upon wine composition

Question 1

What are the 4 sources of flavour and aroma compounds in wine?

Answer 1

• Grape
• From processing- enzymatic reactions.
• Though alcoholic fermentation.
• maturation- chemical reactions through storage.

Question 2

What is the major product of fermentation and what effect in wine flavour and aroma does it have?

Answer 2

Ethanol!

• Warming sensation/heat on the palate.
• ethyl alcohol aroma
• impacts the volatility of other aroma compounds by changing the polarity of the wine matrix.

Question 3

Glycerol is the second major product of alcoholic fermentation. What levels are produced? when is it produced? What is its effect on the palate?

Answer 3

• 2.5 to 14.7 g/l.
• most produced during the early stages of fermentation.
• gives body, mouthfeel and sweetness to the palate. counteracts heat of ethanol.

Question 4

Why is Glycerol produced by the yeast cell?

Answer 4

• As an osmoprotectant in the high sugar environment of early alcoholic fermentation.
• Restores redox imbalance- Replenishes NAD+ that is lost when glycolytic intermediates are lost to amino acid catabolism.

Question 5

How does glycerol act as an osmoprotectant?

Answer 5

Interacts with hydrophobic portions of specific proteins and helps them maintain functional shape and prevent cellular dehydration.

Question 6

How does glycerol correct the redox imbalance arising from the loss of NAD+ from when glycolytic intermediates are lost to amino acid catabolism?

Answer 6

NADH is oxidised to NAD+ as dihydroxyacetone phosphate is reduced to glycerol-3-phosphate. (next step is dephosphorylation to glycerol).

Replenishing NAD+ ensures that glycolysis is able to continue.

Question 7

What are the factors that affect glycerol production?

Answer 7

• Strain variation. some strains are being developed to divert carbohydrate towards glycerol production to produce lower alcohol wines.
• Temperature- higher temps give higher glycerol- up-regulation of glycerol production enzymes.
• Sugar concentration- higher sugar gives higher glycerol due to its functioning as an osmoprotectant.
• Botrytis and moulds- lead to desiccation and higher sugar, therefore, higher glycerol.
• SO2- higher SO2 gives higher glycerol as binding acetaldehyde prevents regeneration of NAD+ via fermentation pathway. Too much SO2 required to be a practical means of increasing glycerol.
• pH- no effect over the range of wine pH.
• Nitrogen- higher glycerol when ammonia is used as sole N source as more glycolytic intermediates are lost to amino acid catabolism.

Question 8

What are the 2 main sources of yeast-derived organic acids?

Answer 8

• from pyruvate via the citric acid cycle- acetic, succinic, a-ketoglutaric, malic and lactic.
• From malonyl CoA though fatty acid synthesis - straight chain fatty acids such as oleic acid.

Question 9

Quantitatively, what is the most significant acid produced by yeast? what pathways can it be produced by?

Answer 9

Sucinnic acid.

Primary oxidative pathway- from glutamate
Minor reductive pathway- from aspartate.

Question 10

Qualitatively, what is the most important acid produced during alcoholic fermentation?

Answer 10

Acetic acid.
1.2-1.4 g/l considered spoiled. Large strain variation and strains producing more than 1 g/l are unsuitable. Other non-saccharomyces strains produce larger quantities (Kloeckera apiculata, Hansenula anomala).

Question 11

Why is acetate produced by yeast?

Answer 11

formation of acetyl CoA for the transfer of acyl groups in fatty acid chain elongation. Formation of sterols and fatty acids for cell membrane.
When acetate production outweighs its incorporation into other compounds it is excreted into wine as acetic acid.

Question 12

What factors affect the production of acetic acid by S. cerevisiae?

Answer 12

• initial sugar conc
• fermentation temp- increase in AA directly proportional to increased biomass production at higher temps.
• nitrogen availability- complicated and depends on nitrogen source.
• pH of juice.- negligible within the range of wine pH.

Question 13

Can yeast degrade malic acid? what to? and to what extent?

Answer 13

• S. cerevisiae can consume and produce malic acid but always produce more than consumption (at wine pH). can degrade up to 45% of available malate.
• Degradation is to ethanol (via pyruvate and acetaldehyde intermediates).
• Schizosaccharomyces (pombe) and Zygosaccharomyces can completely degrade malic acid (maloalcoholic conversion) and is being explored for deacidification.

Question 14

When is pyruvic acid produced and why can it be significant?

Answer 14

• produced under nitrogen limitation as it cannot be converted into other products. Decreases in the presence of nitrogen.
• impacts acid balance and mouthfeel, binds SO2 reducing efficacy.

Question 15

What are the 2 processes that can produce fatty acids in yeast?

Where can the yeast cell use these?

Answer 15

• Anabolic pathway utilising malonyl-CoA. Acetyl-CoA is used for chain initiation. (Main pathway)
• β-oxidation of long-chain fatty acids

Can be incorporated into the cell membrane to assist in ethanol tolerance.

Question 16

What is the sensorial contribution of fatty acids in wine?

Can these form esters with ethanol?

Answer 16

• a positive correlation between medium-chain fatty acids and wine quality.
• may contribute aroma to chenin, colombard and riesling.
• smoothness and roundness to mouthfeel

Yes, these can form esters.

Question 17

What is the relationship between ester formation and primary fermentation rate?
What about oxygen?

Answer 17

Negative relationship. As rate of primary fermentation increases, the rate of ester production decreases. The presence of oxygen increases ester production which is also linked to fermentation rate.

Question 18

What is the most prevalent ester in wine and what rate is it produced at?

Answer 18

Ethyl acetate.

• S.cerevisiae- less than 50 mg/l.
• Non-saccharomyces such as hanseniaspora and pichia can produce much higher levels (up to 900 mg/l)

Greater than 200 mg/l is regarded as spoiled.

Question 19

What are some esters that are beneficial to wine quality? In what way are they beneficial?

Answer 19

Fruity character- iso-amyl acetate, n-hexyl acetate.

Heady aromatics- ethyl octanoate, ethyl decanoate, 2-phenyl acetate.

All produced in much lower amounts than ethyl acetate but have much lower detection thresholds.

Question 20

What can higher alcohols bring to a wine?

Answer 20

Can contribute to flavour and body. Excessive amounts can impair flavour- isoamyl alcohol can produce a burning taste. Red wines contain higher amounts (average 390 ppm) than white wines (310 ppm).

Question 21

How are higher alcohols formed in wine?

Answer 21

1. Amino acids catabolised and converted into higher alcohols. Removal of amine group, carboxylation of the keto acid to an aldehyde and then reduction to a higher alcohol.
2. Anabolic process from pyruvic acid (sugar) metabolism. Utilises acetyl CoA.

Question 22

What factors affect the formation of higher alcohols in wine?

Answer 22

• Yeast strain
• pH- increases with higher pH.
• temperature- different higher alcohols favoured at different temperatures.
• nitrogen- lack of nitrogen leads to higher levels
• oxygen- higher oxygen leads to higher levels.
• Solids- higher suspended solids leads to higher levels. of some higher alcohols.

Question 23

What are the 2 sources of sulfur-containing compounds in wine?

Answer 23

• those naturally present- sulfates, amino acids, proteins.

- Those added to the juice or wine- elemental sulfur, sulfur dioxide.

Question 24

Explain the mechanism of hydrogen sulfide production in yeast

Answer 24

Sulfate reduction sequence

SO4(2-) –>APS–>PAPS–>HSO3(1-)–>H2S + O-AS/O-AH–> Cystein or Methionine.

Sulphate is reduced to sulphide which reacts with O-acetylserine or O-acetylhomoserine (cytosolic nitrogen Pool) to produce cysteine or methionine respectively.

When O-AS and O-AH are in limited supply, the sulphide builds up and diffuses from the cell.

Question 25

How much H2S is produced when sulfate is the source of sulfur and how does this differ when SO2 is the source?

Answer 25

Very little due to feedback mechanisms of the enzymes that convert sulfate to bisulfite. This limits the amount of substrate available to sulfite reductase that produces sulfide. These enzymes re only turned on when sulfur-containing AAs are needed by the cell.

SO2 can freely enter the cell where it forms bisulfite in the less acidic conditions of the cell. This means it bypasses the feedback mechanisms from sulfate to sulfite outlined above. NO FEEDBACK CONTROL.

Question 26

What are the major factors affecting the hydrogen sulphide production during fermentation?

Answer 26

Composition of grape juice-Nitrogen deficiency, deficiency of vitamins, high pH, presence of free sulphur, excessive sulphur dioxide, deficiency of metal cations, presence of grapes solids all increase H2S.

Conditions of fermentation- High fermentation temperature, high vol tanks, yeast strain.

Post fermentation treatment of wine- leaving on lees as this creates strongly reductive conditions.

Question 27

How can the timing of H2S production influence its persistence through to the final wine?

Answer 27

H2S produced early in primary fermentation can be carried away by CO2 by a vigorous fermentation.

That produced near the completion of primary fermentation may be retained through to the final wine.

Question 28

What are the methods for the removal of H2S from wine?

Answer 28

• Aeration (or sparge with nitrogen). Can also remove volatiles. Physically removes it from wine.
• SO2 addition. Binds H2S to form elemental Sulfur. Only works with very low levels.
• Addition of copper ions- forms insoluble copper sulfide. Can be filtered or fined out. Can cause copper-protein hazes. Restrictions on levels in final wine.

Question 29

What is the mechanism by which yeast produce SO2 during fermentation?

Answer 29

From the sulfate reduction sequence. High producers have enhanced activity of sulfite production enzymes and depressed activity of sulfite degrading enzymes.

Low producers can produce 10-30 ppm while high producers can produce over 100 ppm

Question 30

What factors effect SO2 production in yeast?

Answer 30

• deficiency in pantothenic acid increases production
• presence of sulfate
• presence of cysteine and methionine reduces production.
• temperature- high temperatures lead to reduced production.

Question 31

What are mercaptans?
What are their sensory effect?
How can they be removed?

Answer 31

• Sulfur compounds formed through the reaction of ethanol or acetaldehyde with hydrogen sulfide. (Ethyl mercaptan and methyl mercaptan)
• Have a smell of onions/rubber and rotten eggs/cabbage respectively)
• addition of copper can form copper mercaptides and disulphides (which can themselves have a negative aroma).

Question 32

What is DMS?

What is its aroma and what wines is it typically seen in?

Answer 32

Dimethyl sulfide.
Asparagus, cooked corn, black currant. Often seen in late harvest and bottle aged wines.
Cysteine is a precursor for production.

Question 33

What is the flavour profile of acetaldehyde?

What factors influence its production?

Answer 33

Green apple aroma.

High temperatures and oxygen increase production. Formed from sugar metabolism via pyruvate.