Revision questions Flashcards
Explain why some Saccharomyces cerevisiae strains produce SO2 during
fermentation. Include in your answer descriptions of any factors may influence the
production of SO2 by S. cerevisiae.
Modified activities of enzymes associated with the SRS pathway. Most high SO2
producing strains show either enhanced activity of sulphite producing enzymes
or depressed activity of sulphite degrading enzymes.
− Factors which influence the quantity of SO2 produced are:
• A deficiency in pantothenic acid which may cause low SO2 producing yeasts
to increase formation of SO2
• Presence of sulphate enhances SO2 production by high SO2 producers
• Presence of cysteine and methionine reduces SO2 production by both low
and high producers of SO2
• Fermentation temperature – decreased SO2 production at higher
temperatures
Interactions between micro organisms and their biochemical products during
alcoholic fermentation may affect yeast growth. One of the most important
interactions is that between Botrytis cinerea and Saccharomyces cereviseae. Briefly
describe two inhibitory effects of Botrytis infection of grapes and Saccharomyces
growth.
If infection with Botrytis cinerea occurs in wet weather B.cinerea will produce
significant quantities of acetic acid from sugars which has inhibitory effect on
S.cereviseae (sluggish fermentations)
− In hot and dry conditions B.cinerea infection leads to a dessication of the grape
berry and an elevation of the concentration of sugars in the must. Elevated sugar
concentration leads to increased osmotic pressure that places yeast under stress
(increased lag phase to incomplete fermentation with high residual sugars).
S.cereviseae itself also produces more acetic acid in high sugar musts leading to
a loss in wine quality.
Saccharomyces may utilise carbohydrates by respiratory or fermentative
biochemical pathways. Respiration is intrinsically a more efficient means of glucose
catabolism as a greater yield of energy is released compared with the fermentative
pathway. What factors influence Saccharomyces to utilise fermentative pathways in
preference to respiratory pathways and what are the major end products of this
pathway.
Grapes may initially contain a small amount of dissolved oxygen but this is rapidly
consumed and the environment becomes quickly anaerobic. S.cereviseae will
ferment hexose sugars via the glycolytic pathway.
Glucose + 2ADP + 2Pi + 2NAD+ → 2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
S.cereviseae does not metabolise all the glucose it consumes through the glycolytic
pathway and channels part of the sugar through the hexose monophosphate
pathway which does not produce ATP and instead producing NADPH to supply the
reducing power required in many biosynthetic reactions.
List four organic acids produced by yeasts during fermentation?
L-Malic (utilised and formed) Acetic (utilised and formed) Pyruvic (utilised and formed) D-Lactic and Succinic (formed) Citric (neither utilised nor formed)
Describe how ethanol impairs yeast cell function.
Ethanol reduces the efficiency of activated membrane transport systems (either by
increasing membrane permeability to protons or inhibiting the proton pumping
mechanism) and decreases nitrogen and sugar uptake.
Fusel alcohol production by selected yeast strains may vary according to the
fermentation conditions. Briefly describe how the following factors impact fusel
alcohol production.
Yeast strain – production on n-propanol, iso-amyl alcohol, iso-butanol and β-
phenethanol is affected by which strain of S.cereviseae is conducting the
fermentation.
pH – production increases with increasing pH.
Temperature – increases in temperature favour increased production of iso-butanol
and iso-amyl alcohol, whereas decreased production of n-propanol and 2-phenethyl
alcohol.
Nitrogen composition of the must – a lack of amino acids or ammonia will
increase higher alcohol production.
Oxygen – anaerobic conditions decrease higher alcohol production; and
Grape juice solids – the presence of suspended solids in grape juice stimulates the
formation of iso-butanol and iso-amyl alcohol. The production of n-propanol and
active amyl alcohol is not changed significantly.
How can the growth conditions of a yeast starter culture be influenced to maximise
the quantity of sterols and fatty acids available to the yeasts during fermentation?
Tank cultures should always be aerated using sterile-filtered air.
Briefly discuss fermentation parameters that influence the quantity of SO2
production by yeasts.
A deficiency in pantothenic acid which may cause low SO2 producing yeasts
to increase formation of SO2
• Presence of sulphate enhances SO2 production by high SO2 producers
• Presence of cysteine and methionine reduces SO2 production by both low
and high producers of SO2
• Fermentation temperature – decreased SO2 production at higher
temperatures
Outline the biochemical pathways important in the production of H2S by yeasts
during fermentation. Include in you answer an explanation of how these pathways
are regulated by the yeast and the role that yeast assimilable nitrogen has in H2S
production.
Sulfur containing amino acids are produced by yeasts in the sulfate reduction
sequence (SRS). The SRS pathway is a tightly controlled process not normally
active in yeasts unless the concentration of cysteine and methionine in the
fermentation becomes limiting. Under these conditions, the yeast will reduce
inorganic sulphate (SO4
2-) to sulfide (S2-) which is then reacted with the nitrogen
precursors O-AS or O-AH to produce cysteine or methionine respectively. However,
under conditions where the precursors O-AS and O-AH are in limited supply (low
nitrogen conditions) excess sulfide is produced which then diffuses out of the cell as
hydrogen sulfide.
SO2 (added as potassium metabisulfite) rather than sulfate is the major source of
sulfur atoms during H2S production in fermentation. Only minor amounts of H2S are
formed when sulfate is the source of sulfur under nitrogen limiting conditions.
Reactions leading from sulfate to HSO3
- are tightly regulated by feedback inhibition
and are only active when sulfur-containing amino acids are required by the yeast.
This limits the supply of sulfite to enzyme sulfite reductase, which reduces HSO3
- to
suflide. However, SO2 is able to freely enter the cell where it forms HSO3
- that is
then converted to sulfide by sulfite reductase. HSO3
- originating from SO2 bypasses
all the control sites along the SRS so that sulfide is formed without any
feedback control
A number of vitamins are essential enzyme cofactors that participate in the growth of
yeasts during fermentation. Complete the following table that best describes the
most important biochemical pathway or function and the essential vitamin/cofactor.
Biotin − Involved in metabolism of carbohydrates, amino acids, proteins, nucleic acids and lipids − nitrogen metabolism − transport and activation of CO2 in decarboxylation reactions, particularly of pyruvate to oxaloacetate
Inositol
− Important structural function in phospholipids
− influences cell division
Folic Acid
− Important co-factor in biological reactions
involving the transfer of one carbon units.
α-Aminobenzoic Acid (PABA) − Synthesis of folic acid and its related compounds − biosynthesis of pantothenic acid
Vitamin B6 group − phosphorylated and non- phosphorylated compounds are important in microbial metabolism − Pyridoxal-5-phosphate is a co-enzyme in
During fermentation monitoring a winemaker notices a slowing of carbohydrate
utilisation and decides to make an addition of DAP. A 70 ppm addition is made
however no substantial increase in fermentation rate occurs. Explain the nonresponsive
behaviour of the yeast strain to the DAP addition.
Not enough DAP has been added. It is usually added at a rate of 200-500 ppm.
Excessive clarification of juice may arise through employment of highly efficient
technology such as centrifugation. Describe what nutrients are likely to be
decreased by excessive clarification, how these specific nutrients impact
fermentation if not present in sufficient quantities, and what wine production
strategies may be employed to correct deficient nutrient levels.
The removal of grape solids during clarification will also have a major effect on
nutrient availability. Grape solids possess important concentrations of trace
nutrients such as vitamins and sterols which promote the growth of yeasts.
Excessive clarification can cause sluggish fermentations particularly in white wine
where nutrient supplements were minimal.
DAP can be added to supplement the natural levels of nitrogen in must.
During fermentations in which high concentrations of sulphite are present greater
concentrations of glycerol can be produced by Saccharomyces cereviseae. Briefly
describe the biochemical mechanisms responsible for this observation.
Sulfites bind to acetaldehyde preventing its reduction and subsequent regeneration
of NAD+. This occurs by glycerol formation.
Describe the mechanism that allows sulfur dioxide to enter and accumulate in a
yeast cell.
sulfur trap. neutral pH in yeast cell lead to bisulfite ion being formed and more SO2 diffusing into the cell
Complete the net reaction of the glycolytic pathway.
Glucose + 2ADP + 2Pi + 2NAD+ → 2 Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
Which yeast represents the dominant micro flora upon the surface of the grape
berry? Genus and species name is required.
Hanseniaspora uvarum
Describe how the addition of yeast hulls may overcome a stuck or sluggish
fermentation.
They act as a source of essential lipids (sterols). Sterols are a major component of
the cell membrane and play an important role in ethanol tolerance of the organism.
Also, absorb medium-chain fatty acids which inhibit fermentation.
List three nitrogen containing compounds in grape must.
- Ammonium ions
- Amino acids
- Peptides and proteins
Which two transport systems are important for amino acid absorption during
fermentation?
- Specific amino acid transporter complexes, and/or
- A group-specific transporter system called the general amino acid permease
(GAP)
Briefly outline the effects of wine pH upon yeast amino acid absorption during
fermentation.
The pH optima for ammonium absorption is 6 to 6.5, indicating that the uptake
efficiency of this ion is reduced at wine pH. However, the uptake efficiency appears
to be unaffected at juice and wine pH.
What is the major role of pantothenic acid in yeast metabolism?
It is part of co-enzyme A (CoA). CoA is involved in the acetylation of amino acids,
glucosamine, hydroxyalanine, choline, sulfaniliamide and histamine. CoA is also
involved in the formation of fatty acids from acetate and may also be involved in the
synthesis of biotin.
Outline the formation of fusel alcohols (higher alcohols) from amino acid
metabolism.
The carbon skeletons of some amino acids are catabolised and converted into
alcohols. This involves the removal of an amino group by deamination followed by
decarboxylation of the resulting keto acid to an aldehyde. The aldehyde is then
reduced to form a higher alcohol with one less carbon than the original amino acid.
Describe the contribution of fusel alcohols to wine quality.
Higher alcohols contribute partially to the flavour and body of wines, when present in
excess amounts they impair flavour of the wine eg excessive contributions of isoamyl
alcohol produces a burning taste in wine. However, 2-phenethyl alcohol
imparts a ‘rose’ aroma to wine.
Describe the two major sources of sulfur containing compounds in wine and provide
examples of each.
Compounds naturally present in the juice – including sulphates, amino acids, or
proteins containing these amino acids, and
Those added to the juice or wine – a winemaker may add sulfur or sulfur
containing compounds before harvest (eg elemental sulfur on the grapes as an antifungal
agent), or at crushing, during or post fermentation (potassium metabisulfite
which forms SO2 in the juice or wine).
Briefly describe how temperature during fermentation may influence yeast growth,
replication and subsequent wine quality.
Temperature influences growth of individual species. The growth on non-
Saccharomyces yeasts is stronger at lower temperatures (10oC to 15oC). This will
have an indirect effect on the sensory properties of wines since those yeasts
produce significant aroma active compounds such as esters, higher alcohols and
acetic acid. Additionally, these yeasts decrease the amount of sugars available for
consumption by S.cereviseae leading to wines with reduced ethanol content.
Clarification prior to fermentation will result in removal of some nutrients from grape
juice. List three specific nutrients of grape juice that can be affected by clarification.
Vitamins, unsaturated fatty acids and sterols in grape solids
