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1

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.

The reason why some yeasts are high producers of SO2 is believed to be due to modified activities of enzymes associated with the SRS pathway. A deficiency in pantothenic acid which may cause low SO2-producing yeasts to increase formation of SO2.

2

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.

Botrytis infection causes increased glycerol in finished wines partially due to increased sugar concentration of grapes. Botrytis cinerea also produces an antimicrobial substance called Botryticine during growth on grapes that alters yeast carbon flux.

3

List four organic acids produced by yeasts during fermentation.?

Succinic, acetic, malic, and pyruvic acids.

4

Describe how ethanol impairs yeast cell function.

Ethanol, a major product of the alcoholic fermentation, exerts a toxic effect on yeast principally through a reduction in sugar and nitrogen transport across the cell membrane.

5

Fusel alcohol production by selected yeast strains may vary according to the fermentation conditions. Briefly describe how the following factors impact fusel alcohol production.

During the alcoholic fermentation, yeasts will produce small concentrations of higher alcohols, principally n-propanol. Although higher alcohols may contribute partially to the flavour and body to wines, it is generally believed that when present in excess amounts they impair the flavour of wine.

6

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?

Two major groups chemical additions to the juice, and clarification procedures.
1. Added SO2 will increase lag phase. Added nutrient supplements such as (DAP) helps with limited nitrogen concentrations. Vitamins supplements and yeast hulls which absorb medium chain fatty acids and act as a source of essential lipids (Sterols) which play an important role in ethanol tolerance which may overcome stuck ferments.


7

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?

Two major groups chemical additions to the juice, and clarification procedures.
1. Added SO2 will increase lag phase. Added nutrient supplements such as (DAP) helps with limited nitrogen concentrations. Vitamins supplements and yeast hulls which absorb medium chain fatty acids and act as a source of essential lipids (Sterols) which play an important role in ethanol tolerance which may overcome stuck ferments.


8

Briefly discuss fermentation parameters that influence the quantity of SO2 production by yeasts.

SO2 produced will become bound to other yeast metabolites (such as acetaldehyde), thereby increasing the total SO2 content of the wine. For wines already containing high levels of total SO2, it is possible to actually exceed the legal limit. Wines with excessive levels of bound SO2 may also develop a bitter taste.


9

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.


Hydrogen sulfide is produced by yeasts during fermentation during synthesis of sulfur­ containing amino acids. The most studied biochemical pathway is the sulfate reduction sequence (SRS)
The SRS pathway is a tightly controlled process that is normally not active in yeast unless the concentration of cysteine and methionine in the fermentation becomes limiting.


10

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
Biotin is involved in a wide variety of metabolic processes including the metabolism of carbohydrates, amino acids, proteins, nucleic acids and lipids (Figure 5).  Biotin also plays an essential role in nitrogen metabolism and in decarboxylation reactions. 
Inositol
Inositol (meso-inositol, myo-inositol, i-inositol) is stimulatory or essential for growth of a number of yeasts (Figure 7).  The actual role of inositol in cell metabolism is not clear, although it seems to be involved in the actual structure of the cell through its presence in phospholipids (particularly in cell membranes).
Folic Acid
Folic acid, (as tetrahydrofolate), is involved in the metabolism of one-carbon fragments, such as transmethylation.  Myo-inositol serves a structural function in phospholipids.
p-Aminobenzoic Acid
The main function of PABA (Figure 11) appears to be in the synthesis of folic acid and its related compounds
Vitamin B6 Group
All vitamins needed by yeasts (with the exception of inositol) serve vital catalytic functions as part of co-enzymes in the metabolism of yeasts. 
Riboflavin
The importance of riboflavin (Figure 13) lies in the co-enzyme derivatives, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).  Free riboflavin has no known co-enzymic function.  FAD is[…]”

11

What is Biotin?

Biotin is involved in a wide variety of metabolic processes including the metabolism of carbohydrates, amino acids, proteins, nucleic acids and lipids.  Biotin also plays an essential role in nitrogen metabolism and in decarboxylation reactions.

12

What is Inositol?

Inositol (meso-inositol, myo-inositol, i-inositol) is stimulatory or essential for growth of a number of yeasts. The actual role of inositol in cell metabolism is not clear, although it seems to be involved in the actual structure of the cell through its presence in phospholipids (particularly in cell

13

What is Folic Acid?

Folic acid, (as tetrahydrofolate), is involved in the metabolism of one-carbon fragments, such as transmethylation.  Myo-inositol serves a structural function in phospholipids.


14

What is p-Aminobenzoic Acid?

The main function of PABA (Figure 11) appears to be in the synthesis of folic acid and its related compounds

15

What is the Vitamin B6 Group?

All vitamins needed by yeasts (with the exception of inositol) serve vital catalytic functions as part of co-enzymes in the metabolism of yeasts. 

16

What is Riboflavin?

The importance of riboflavin (Figure 13) lies in the co-enzyme derivatives, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).  Free riboflavin has no known co-enzymic function.  FAD is important for the proper functioning of the respiratory chain, and is involved in the transfer of hydrogen or electrons from the reduced form of NAD to cytochromes.


17

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 non-responsive behaviour of the yeast strain to the DAP addition.

Yeasts ferment at a slower rate under nitrogen-starvation conditions because they lack the necessary nitrogen with which they can synthesise new proteins for growth (e.g. enzymes). A major difficulty facing winemakers is that fermentations that become sluggish due to nitrogen-starvation may potentially become non-responsive to the addition of nitrogen supplements such as DAP.

18

A white grape juice must at 20°C is inoculated with 1% volume of a wet yeast starter culture which has a total yeast cell count of 4.2x108 cells/mL. Briefly describe the growth phases of the yeast inoculum and the chemical changes in the must in respect to alcohol and sugar concentration that will occur during growth. Assume the juice has been pasteurised and that no endogenous viable yeast is present prior to inoculation.

1.Short lag phase 2- 5 days.
2.Followed by a stationary phase where number of viable cells may decreaseFermentative activity (Ethanol production) reaches maximum during this stage.
3.Composition of wine becomes inhibitory due to rising ethanol concentrations. Yeast enters a decline phase and consumption of sugars and ethanol production and carbon dioxide is slowed​


19

Briefly describe two mechanisms for the production of fusel alcohols by yeasts during fermentation.

1.The carbon skeletons of some amino acids are catabolised and converted into higher alcohols.
2. Higher alcohols may also be produced via anabolic processes originating from pyruvic acid (sugar) metabolism .


20

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.


Leads to low solids content in must like sterols and fatty acids which support yeast growth.​


21

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.

Glycerol is also produced by Saccharomyces cerevisiae during biomass formation at commencement of fermentation to protect the cells from the high osmolar concentrations of sugars.

22

Describe the mechanism that allows sulfur dioxide to enter and accumulate in a yeast cell.

The presence of sulfur dioxide during fermentation will cause increased glycerol formation as sulphites bind to acetaldehyde preventing its reduction and subsequent regeneration of NAD+. Regeneration of NAD+ occurs by glycerol formation.

23

Explain the mechanism by which the addition of diammonium phosphate (DAP) decreases production of hydrogen sulphide by yeasts during primary fermentation.

The mechanism explaining the non-responsive behaviour of S. cerevisiae to nitrogen supplementation relates to the activity of transport systems responsible for bringing glucose into the cell. A lack of nitrogen appears to trigger an irreversible arrest of protein synthesis in the cell, which explains why the addition of DAP after this event has little effect in stimulating sugar catabolism in stuck fermentations.

24

List three organic acids produced by yeasts during fermentation besides succinic acid.

Malic, Acetic, Pyruvic

25

Complete the net reaction of the glycolytic pathway.

Glucose + ADP + + 2Pi + 2NAD+​
 2 Pyruvate + ATP + + + 2H2O ​
 2 NADH + 2H+ + 2H2O
Pyruvate that is produced during glycolysis is then decarboxylated to acetaldehyde (thereby producing carbon dioxide), which in turn is converted to ethanol

26

Describe the changes in yeast membrane composition that arises from aeration of must at the beginning of yeast cell growth. What are the physiological advantages for yeast cells that arise from these membrane changes? ​


Yeasts require oxygen in order to produce a variety of sterols and unsaturated long chain (C16 and greater) fatty acids. These compounds are essential components of the cell membrane and are involved in the maintenance of structural integrity and functionality. Under aerobic conditions, S. cerevisiae can manufacture sterols and fatty acids from acetyl-CoA

27

List the three major sources of yeasts within a winery.

On the grape, in the winery and in the must (Starter Culture).​


28

Which yeast represents the dominant micro flora upon the surface of the grape berry? Genus and species name is required.

Hanseniaspora uvarum (and its anamorph, Kloeckera apiculata)

29

Provide four examples of intrinsic factors of grape must that affect growth of yeast during alcoholic fermentation. ​


Concentration of essential nutrients, pH, dissolved oxygen, fungicides, insoluble solids

30

Describe how the addition of yeast hulls may overcome a stuck or sluggish fermentation. ​


1 They adsorb toxic yeast byproducts such as medium chain fatty acids (C8:0 and C10:0)
2. They act as a source of essential lipids (sterols). Sterols are a component of the cell membrane and play an important role in ethanol tolerance of the organism.


31

Addition of sulphur dioxide to juice to minimise juice oxidation is a common practise. Describe how this may influence yeast populations and subsequent growth in the juice.

The toxic effects of sulfur dioxide in yeasts relate to its action against various intracellular enzymatic functions and nucleic acid chemistry.Inside the cell, sulfite may impact upon cell metabolism through:
• cleaving disulfide bridges in proteins (sulfitolysis), thereby reducing the functionality of the molecule;
• destruction of thiamine and co-factors such as NAD+, FAD+, and FMN;
• exhaustion of cellular ATP levels by inhibition of enzymes of the glycolytic pathway (e.g. alcohol dehydrogenase), particularly below pH 4.0;
• transformation of the nuclear base cytosine to uracil, and binds to uracil and thymidine;
• cross-linking between proteins and nucleic acids.

32

List three nitrogen containing compounds in grape must.

Ammonium Ions, Amino Acids, Peptides, Proteins​


33

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).

34

What are two deleterious effects that DAP additions may have upon nitrogen metabolism during fermentation.

Fermentation becomes sluggish due to nitrogen starvartion.A lack of nitrogen appears to trigger an irreversible arrest of protein synthesis in the cell, which explains why the addition of DAP after this event has little effect in stimulating sugar catabolism in stuck fermentations.

35

Briefly outline the effects of wine pH upon yeast amino acid absorption during fermentation.

“Within the range of 3.2 4.0 the effect of pH on acetic acid production is negligable.

36

What is the major role of pantothenic acid in yeast metabolism?

Pantothenic acid is an integral part of the molecule co-enzyme A (CoA). CoA is the major pantothenic acid containing compound in most microorganisms.


37

Outline the formation of fusel alcohols (higher alcohols) from amino acid metabolism. ​


Selected amino acids, and the corresponding keto acids and higher alcohols (the first four form the major components in the higher alcohol component of wines).
Higher alcohols may also be produced via anabolic processes originating from pyruvic acid (sugar) metabolism.

38

Describe the contribution of fusel alcohols to wine quality. ​


Higher alcohols may contribute partially to the flavour and body to wines, it is generally believed that when present in excess amounts they impair the flavour of wine.

39

Describe the two major sources of sulfur containing compounds in wine and provide examples of each. ​


Compounds naturally present in the juice - these include sulfates, 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 (elemental sulfur on the grapes as an anti-fungal agent), or at crushing, during fermentation and post fermentation.

40

Briefly outline the role of cysteine, methionine and nitrogen in relation to H2S production by yeasts during fermentation.

The presence of cysteine and methionine reduces​
SO2 production by both low and high producers of SO2.nitrogen is an important factor in controlling H2S production for most yeast.

41

Briefly describe how temperature during fermentation may influence yeast growth, replication and subsequent wine quality.

Temperature influences growth so increasing temperature (up to 30c) affects the primary fermenation by raising its rate reaction. Over 40c yeast viability is affected and ferment may become stuck. Low temperature ferments tend to preserve the fruity aroma of the grapes, while higher temperatures produce a wine with more subtle fruit flavours and increased extraction of colour compounds from red grape skins.

42

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.

Sterols , Fatty Acids , Nitrogen and Vitamins​


43

How will the removal of indigenous yeast strains by clarification impact wine quality.

Clarification may be achieved by a number of treatments including coldsettling, enzyme addition, centrifugation, bentonite fining, and filtration. Some of these procedures such as centrifugation and filtration affect yeast growth by physically removing indigenous yeast populations. Other treatments such as coldsettling will favour yeasts more adapted to growth at low temperatures.

44

Glucose is channelled through glycolysis and a second major biochemical pathway in yeasts. Name and describe the function of the other biochemical pathway involved in the degradation of glucose by Saccharomyces. ​


Hexose Monophosphate Pathway - does not produce ATP to drive energy - expensive reactions but NADPH that is used to supply the reducing power required in many biosynthetic reactions.

45

A winemaker makes an addition of 250 ppm DAP to a red grape must immediately prior to addition of a starter culture of a Saccharomyces sp. Describe how the addition of DAP will influence the uptake and utilisation of grape amino acids by the inoculated starter culture. ​


Diammonium phosphate (DAP) which is added to juice for alleviation of difficulties associated with limiting nitrogen concentrations. These problems include slow or stuck fermentations and/or H2 S production.

46

Discuss the impact of ethanol concentrations and nitrogen absorption by yeast during alcoholic fermentation. What impact does the timing of nitrogen absorption by yeasts have upon ferment management?

Ethanol, a major product of the alcoholic fermentation, exerts a toxic effect on yeast principally through a reduction in sugar and nitrogen transport across the cell membrane.

47

During anaerobic conditions yeasts lack the ability to produce long chain unsaturated fatty acids and sterols and must therefore obtain these as preformed nutrients. What sources of fatty acids and sterols are available for yeasts in ferments that have not been supplemented with vitamins or yeast hulls by the winemaker?

The reserves of sterols and fatty acids built up prior to the alcoholic fermentation (as during starter culture preparation the presence of lipid-containing material in the juice from which they can scavenge the necessary unsaturated fatty acids in order to synthesize the required sterols (grape skins is one such source).

48

Succinic acid is formed by reductive and oxidative processes during fermentation. What are the precursors for each metabolic pathway and describe the overall contribution of succinic acid to wine quality. ​
Oxidative pathway precursor Reductive pathway precursor Contribution to wine quality .

The most significant acid produced during fermentation is succinic acid, which may be produced via two major pathways: (i) an oxidative pathway and (ii) a minor reductive pathway, both involving enzymes of the citric acid cycle

49

Explain why some strains of Saccharomyces cerevisiae produce SO2 during fermentation. What conditions of fermentation and grape/wine constituents influence SO2 production? ​


Yeasts are able to produce varying quantities of SO2 during fermentation. This is an important characteristic in winemaking yeasts because the SO2 produced will become bound to other yeast metabolites (such as acetaldehyde), thereby increasing the total SO2 content of the wine. The reason why some yeasts are high producers of SO2 is believed to be due to modified activities of enzymes associated with the SRS pathway.

50

Describe how the composition of grape juice will influence H2S production by yeasts during fermentation. ​


Hydrogen sulfide is produced by yeasts during fermentation during synthesis of sulfur containing amino acids. The most studied biochemical pathway is the sulfate reduction sequence (SRS)

51

Succinic acid is the most abundant organic acid produced by yeasts during fermentation. What other important organic acids are produced by yeasts during alcoholic fermentation? ​


Malic, Acetic, Pyruvic acids are formed.

52

Describe the mechanisms and any inhibitors of glucose and fructose uptake by Saccharomyces cerevisiae during primary (alcoholic) fermentation. ​


A low affinity for glucose and fructose, and another system which has a high affinity for these sugars. During the growth phase of fermentation, the low affinity carrier system is expressed constitutively, although during the stationary phase it decreases in activity. The high affinity carrier system is inactive during the early stages of fermentation as it is repressed by the high concentrations of glucose present in the juice.Activity in this transport system remains low until the sugars in the wine are almost exhausted.

53

Amino acid analysis of a shiraz must prior to yeast inoculation showed that the predominant amino acids in descending order are arginine, proline, threonine and glutamic acid. How might the uptake of these amino acids by Saccharomyces cerevisiae vary in the presence and absence of ammonium ions. ​


The pattern of uptake of amino acids during fermentation is largely dependent on the available nitrogen compounds and the genetic preferences of the strain. Ammonium delays the uptake and complete utilisation of Groups A and B amino acids Note that the presence of ammonium ions also suppresses the activity of the GAP (GENERAL AMINO ACID PERMEASE)system.

54

Commencing with pyruvate draw the chemical reactions that occur during the final stages of alcoholic fermentation. Include co-factors where required. ​


Pyruvate that is produced during glycolysis is then decarboxylated to acetaldehyde (thereby producing carbon dioxide), which in turn is converted to ethanol

55

List the three major sources of yeast within a winery.

On the grape, On the machinery and equipment in the winery, in the starter culture in the must.​


56

How can the growth conditions of a yeast starter culture can be managed to maximise the quantity of sterols and fatty acids available to the yeasts during the final stages of fermentation? ​


Ergosterol should be considered an indispensable growth factor when the yeasts are growing under anaerobiosis, since under these conditions they are incapable of synthesising sterols. Skins floating or held immersed within a fermenting juice activated anaerobic fermentation.  It was noted that grape seeds also stimulated anaerobic fermentation, although not to the extent of the berry skins

57

Succinic acid is formed by reductive and oxidative processes during fermentation. What are the precursors for each metabolic pathway and describe the overall contribution of succinic acid to wine quality. ​


Succinic acid, which may be produced via two major pathways: (i) an oxidative pathway and (ii) a minor reductive pathway, both involving enzymes of the citric acid cycle. The major impact of succinic acid on the sensory properties of wine is its contribution to the overall acidity of the product.

58

During fermentation a number of inhibitory substances are produced by some yeast species. List three of these substances. ​


Ethanol, Acetic acid, Killer Toxins

59

List four (4) desirable fermentation properties of a yeast starter culture. ​


Rapid fermentation, High osmotolerance, high ethanol tolerance, effecient conversion of sugars.

60

List three (3) advantages of using selected yeast strains to initiate fermentation rather than indigenous yeasts for spontaneous fermentation. ​


Improved predictability, low production sulfur compounds, high production of glycerol

61

Describe how rehydration of ADWY at low temperatures may cause a loss in viability of the yeast cells. ​


Rehydration at lower temperatures results in excessive loss of cytoplasmic contents before membrane function is re-established. Yeasts that are damaged in this manner will exhibit poor viability and fermentative activity.

62

Excessive growth of which organism is mostly responsible for ester taint? Genus and species name required. ​


The contribution of esters to wine aroma is profound. The most prevalent ester in wines is ethyl acetate

63

Commercial preparation of active dried wine yeast (ADWY) starter cultures involves growth of the yeast in conditions conducive to good fermentation performance following rehydration and inoculation of juice. List four (4) fermentation performance indicators of ADWY cultures and describe how the preparation and dehydration of the starter culture may influence the fermentation characteristic. ​


Rapid initiation of fermentation, high osmotolorance, efficient conversion of sugars to ethanol, high ethanol tolerance. Preparation has to be done with water at 40c as anything lower could cause rexcessive loss of cytoplasmic contents.

64

Describe three (3) adverse outcomes that may arise from the continuous topping of wet yeast starter cultures. ​


Limited nutrient levels in culture, contamination (i.e. bacteria) Genetic make up may change from low H2S producers to high H2S producers.

65

Describe how an initially high sugar concentration in juice may affect yeast growth and metabolism.

High glycerol production and hight acetic acid can be produced

66

What is the most significant advantage in using a selected yeast strain with K2 killer activity? ​


K2 + yeasts are resistant to wild yeast killer activity ​


67

Describe the mechanisms of sulfur dioxide toxicity to yeast growth and metabolism when present in high concentrations within juice. ​


The toxic effects of sulfur dioxide in yeasts relate to its action against various intracellular enzymatic functions and nucleic acid chemistry. Sulfur dioxide (SO2) is able to freely diffuse across the cell membrane whereupon it forms the bisulfite ion (HSO3-) due to the near-neutral pH environment inside the yeast.

68

Excessive clarification of white juice may lead to sluggish and/or stuck fermentations. Briefly describe why this may arise. ​


Leads to low solids in the must and removes a lot of the nutrients such as sterols and unsaturated fatty acids.

69

Name three (3) genera of yeasts that are commonly associated with surface film formation? ​


Candida, Metschnikowia and Pichia

70

Zygosaccharomyces bailii is sometimes associated with refermentation of packaged wines. What properties of this yeast enable survival and growth in finished packaged goods?

The organism survives within the concentrate which due to high osmotic pressure causes the cells to significantly reduce in size.

71

Name the genus of yeast that may completely degrade malic acid to ethanol but which may also cause development of off flavours. ​


S. pombe and S. malidervorans

72

List four (4) desirable production properties of a yeast starter culture. ​


Rapid initiation of fermentation,high osmotolerance (to sugars); ​efficient conversion of sugars to ethanol; ​high ethanol tolerance

73

Write concise notes on stuck or sluggish fermentations due to the presence of high sugar levels in grape juice/must. ​


Sugar concentration will exert a selective effect (osmotic shock) on the strains of yeast that conduct the fermentation. The occurrence of sluggish ferments can easily be detected by a reduction in the rate of sugar consumption. Yeasts ferment at a slower rate under nitrogen-starvation conditions because they lack the necessary nitrogen with which they can synthesise new proteins for growth (e.g. enzymes).

74

List four (4) desirable sensory characteristics of a yeast starter culture. ​


Low production of volatile acidity, acetaldehyde, and higher alcohols;
low production of sulfur compounds (H2S, thiols, DMS); ​
high production of glycerol; ​
enhanced autolytic and glycosidase activity6; ​
modified esterase activity. ​


75

Describe the effects on an ADWY that is rehydrated at 40°C for 15 minutes and is then rapidly transferred to a clarified Riesling must held at 14°C.

Can be destructive to vitamins, down regulated enzyme activity and less glycerol is produced.​


76

How may the addition of yeast hulls be beneficial for preventing incomplete fermentation?

1. They adsorb toxic yeast byproducts such as medium chain fatty acids (C8:0 and C10:0)
2. They act as a source of essential lipids (sterols). Sterols are a component of the cell membrane and play an important role in ethanol tolerance of the organism. Providing yeast hulls may help to overcome a stuck or sluggish fermentation.

77

Describe how the presence of sulfur dioxide (SO2) in juice/wine may lead to an increased concentration of sulfite (SO3-) in yeast cells. ​


Sulfur dioxide (SO2) is able to freely diffuse across the cell membrane whereupon it forms the bisulfite ion (HSO3-) due to the near-neutral pH environment inside the yeast.

78

Describe the antimicrobial actions of sulfite with respect to yeast cells. ​


SO2 is added mainly to minimise oxidation of the juice, it also impacts on the yeast population due to its antimicrobial properties.
SO2 will increase the length of the lag phase, the time for completion of fermentation, and the death rate of the yeast population following sugar exhaustion.

79

Write concisely about wine spoilage caused by Pichia yeasts.

The organism forms folded films over the surface of the wine during conservation and produces significant concentrations of acetic acid (and its esters) and acetaldehyde. Its ability to tolerate relatively high levels of ethanol (up to 14% v/v) and sulfur dioxide (up to 250 - 500 ppm total) are the major factors responsible for its presence in wine after fermentation .​


80

Describe the typical sensory characters of a wine spoiled through the growth of Dekkera yeast. ​


Haze formation, by production of excessive amounts of acetic acid and esters.

81

Which yeast has been associated with the production of a mousy off flavour in wine?

Dekkera has been implicated in the formation of other unpleasant off-flavour compounds such as an apple or cider-like aroma, ‘mousy-off-flavour’ (substituted tetrahydropyridine compounds) and phenolic taints

82

In the preparation of active dried wine yeast (ADWY) starter cultures production strategies are employed to ensure the yeast strain has desirable fermentation performance characteristics. Describe how ADWY are prepared to ensure the following performance indicators are achieved.

An important step in ensuring high viability in reactivated dried yeast cultures is rehydration with warm water at about 40°C. Utilising warm water maximises the chances of successful re-establishment of functionality of the cell membrane. Rehydration at lower temperatures results in excessive loss of cytoplasmic contents before membrane function is re-established. Yeasts that are damaged in this manner will exhibit poor viability and fermentative activity.
Following successful rehydration, the cells should be quickly transferred to the must otherwise they will rapidly exhaust their intracellular nutrient reserves.

83

List four desirable sensory characteristics of a yeast culture used for wine production. ​


Low production of volatile acidity, acetaldehyde, and higher alcohols;
Low production of sulfur compounds (H2S, thiols, DMS); • high production of glycerol;
enhanced autolytic and glycosidase activity

84

List three compounds that may be present in grape juice and/or must that may be inhibitory to yeast growth. Describe the inhibitory mode of action on yeast growth for each of these compounds.

Ethanol - toxic properties that disrupt membrane functionality
Acetic Acid - Acetic acid in high concentration will cause yeast cytoplasmic pH to decrease which disrupts glycolytic enzyme function
H2S - gives a foul odour (rotten egg) and can react with other materials in the wine and form mercaptan.

85

What are survival factors and how do they influence alcoholic fermentation? ​


The ability of yeast hulls to stimulate yeast growth during sluggish fermentations through these mechanisms has led it to be termed a survival factor. The presence of long chain fatty acids with a high degree of unsaturation appears to aid S. cerevisiae to tolerate ethanol. Chemical analysis has shown that yeast hulls contain a large quantity of sterols and unsaturated fatty acids. As indicated earlier (and in Topic 2), sterols and unsaturated fatty acids are important components of the cell membrane, and assist in conferring ethanol tolerance to yeasts.

86

List five advantages and five disadvantages of the use of active dried yeast cultures for inoculations of must or juice to make wine.

Advantages - Rapid ferment, high osmotolerance, efficient conversion of sugars to ethanol, high ethanol tolerance, fermet at low temperature (white wine)

87

Describe the mechanisms of sulfur dioxide toxicity to yeasts. ​


The SRS pathway is a tightly controlled process that is normally not active in yeast unless the concentration of cysteine and methionine in the fermentation becomes limiting.

88

Describe the processes occurring that comprise the sulfite trap. What effect does the sulfite trap have upon yeast metabolism? ​


Transformation of SO2 to SO3- encourages more SO2 to diffuse into the cell until an equilibrium is established between the exterior and interior of the cell (the so called sulfite trap mechanism).

89

Wine spoilage may be caused by Zygosaccharomyces bailii. What properties of this yeast enable growth in finished wine products? ​


The organism survives within the concentrate which due to high osmotic pressure causes the cells to significantly reduce in size. Subsequent re-hydration of the cells to their physiological size and state, once placed into the finished product, takes some time to be fully complete and during this time passage of the cells through 0.45μm pore size membranes is possible

90

Which yeasts (genus and species) are most likely to be responsible for production of ester taint in wine? Describe control measures that will minimise this fault during wine production.

Hanseniaspora uvarum (and its anamorph Kloeckera apiculata), Metschnikowia pulcherrima, Pichia anomala and yeasts belonging to the genera Dekkera.
Minimising fruit damage so that their growth is limited prior to the fermentation
Clarification of the juice (cold-settling, enzyme treatment, addition of bentonite etc.) so that suspended solids are reduced (thereby removing a significant component of the indigenous yeast population); and
Inoculation with a high concentration of the selected yeast strain establishes a large and healthy population of the selected starter culture which rapidly initiates ethanol production.

91

To effectively control of yeast growth in wines what range of molecular sulfur dioxide should be maintained?

Sulphur levels between 0.5 - 0.8 mg/L has been found to be useful in controlling Dekkera/Brettanomyces.

92

Describe how a wet yeast starter culture should be bulked up prior to use as inoculum for fermentation. Provide in your answer the physical and chemical parameters that should be monitored and target specifications.

Preparation of ‘wet’ starter cultures involves propagating the culture up through successive stages (in increasingly larger tanks) until sufficient yeast numbers are available for inoculation into must. In the final stage of yeast propagation, the starter inoculum is usually about 1 - 3% of the final volume of the must (vol/vol).

93

What is the advantage of inoculating juice or must with a known K2 killer toxin active strain of yeast? ​


Using killer yeast in wine fermentations was to suppress the growth of wild yeast, particularly the non-Saccharomyces yeasts. It is probably more useful to use killer strains of S. cerevisiae as a means of resistance to wild strains, rather than for its ability to kill them.

94

Name 2 compounds produced by growth of Dekkera yeast that result in diminished wine quality.

Causes spoilage in wine , as in refermentation in bottled wine causing haze and by products such as acetic acid and esters. Also cause off flavours or mousy or apple cider aromas and phenolic taints.​


95

Describe how each of the following compounds exerts an inhibitory effect on yeast growth. ​


Ethanol - exerts a toxic effect on yeast principally through a reduction in sugar and nitrogen transport across the cell membrane.
Acetic Acid - Acetic acid in high concentration will cause yeast cytoplasmic pH to decrease which disrupts glycolytic enzyme function
High Sugar Levels - Higher Osmotic pressure on yeasts​


96

Describe the outcome that may occur to a fermentation that has been inoculated with an improperly re-hydrated yeast starter culture. ​


Rehydration at lower temperatures results in excessive loss of cytoplasmic contents before membrane function is re-established. Yeasts that are damaged in this manner will exhibit poor viability and fermentative activity.

97

The addition of yeast hulls to a fermenting juice may be useful in certain situations. What are the main nutrients derived from the use of yeast hulls and why should they be added to the ferment? ​


They act as a source of essential lipids (sterols). Sterols are a component of the cell membrane and play an important role in ethanol tolerance of the organism. Providing yeast hulls may help to overcome a stuck or sluggish fermentation.

98

Describe six desirable properties of a starter culture used for inoculation of malolactic fermentation. ​


1.Rapid initiation of fermentation;
2.High osmotolerance (to sugars);
3.Efficient conversion of sugars to ethanol;
4.High ethanol tolerance
5.Able to ferment efficiently at low temperature (white wines).
6.Low production of sulfur compounds (H2S, thiols, DMS);
high production of glycerol