Module 5 - Analysis Flashcards
(128 cards)
1
Q
How do you correct a hydrometer reading for temperature?
A
Measure temperature and perform correction
T1 = Measured sample temperature
T2 = Hydrometer calibratrion temperature
Corrected hydrometer reading = Hydrometer reading (Be) + [0.05 x (T1 - T2)]
Convert to Brix using : Brix = Be x 1.80
2
Q
What is refractometry?
How does a refractometer work?
A
Measure of refractive index: how much light bends in passing from air into solution.
Light enters, passes through sample and into a prism.
If we have a low sugar concentration, light will bend a limited amount and will be projected onto a scale (e.g., Brix).
High concentration much more bending of the light and a high portion of the scale will be lit up.
3
Q
How does glucose impact refractive index in a sample?
A
As we increase glucose concentration refractive index changes in a linear manner.
4
Q
How do we convert Brix to Baume?
How do we convert from Baume to Brix?
A
Convert to Baume: Be = Brix / 1.80
Convert to Brix: Brix = Be x 1.80
5
Q
How do you calculate pH?
A
pH is equal to -log [H+]
- Where [H+] is in mole per litre (mol/L0
- Because H+ has an atomic mass of 1/018 g/mol
- 1 mol/L H+ = 1 g/L H+ or 1.018 g/L to be exact
6
Q
How do you measure pH?
A
Use pH electrode and meter ensuring it is meticulous care and maintenance.
Must be calibrated properly.
7
Q
What is titratable acidity?
A
- Measurement of free and undissociated H+ and a better measure of perceived acidity on the palate?
Explanation:
- In wine we have weak organic acids (e.g., tartaric acid). Acids donate hydrogen ions.
- Some hydrogens are not acidic (e.g., directly to carbon or oxygen attached to a carbon). Some hydrogens are acidic (e.g., those attached to carboxylic acid units).
- Weak acids (e.g. tartaric acid) don’t release all their acidic hydrogens.
- Three forms of tartaric acid in equilibrium: tartrate which has lost all of it’s acidic hydrogens, hydrogen tartrate which has lost one of its acidic hydrogens, and tartaric acid which has lost none of its acidic hydrogens.
- Weak acids generally are distributed amongst different forms (equilibirum forms).
- When we measure pH we are just measuring hydrogen ions that have been released. Not the acidic hydrogens not released.
- TA we are measuring all the acidic hydrogen ions.
8
Q
How do we work out the distribution of forms of tartaric acid at a given pH value?
A
Published pKa values allow us to work out distribution of forms at a given pH value.
At pKa1 = 2.98 we have equal amount of tartaric acid (H2T) and Hydrogen Tartrate (HT-)
At pKa2 = 4.34 we have equal amonts of HT- and Tartrate (T2-).
9
Q
What is the distribution of tartaric acid forms in wine?
A
In very acidic conditions tartaric acid dominates.
At pH 3 we have a similar portion of tartaric acid and hydrogen tartrate.
As we get to higher pH above 4.5 we get tartrate dominating.
10
Q
What happens to wine when it hits our palette?
A
Palate / saliva is pH of 6 so hydrogen ions released from tartaric acid forms and hydrogen tartrate forms becoming tartrate.
Perceived acidity on the palate related better to titratable acidity than pH.
11
Q
Why is pH important for wine stability?
A
Important for wine stability:
- influences wine oxidation
- influences microbial spoilage
12
Q
What is the end point of titration when measuring TA?
A
Standard pH chosen for end point: 8.2
TA value should be qualified by a statement of the end point standard (e.g., TA of 8GL-1 tartaric acid equivalents at pH 8.2)
13
Q
How is Titratable Acidity measured?
A
- Determination of TA by performing titration
Amount of base (NaOH) which is required to react with free H+ and acidic bound H+ (undissociated H+)
Amount of base used is assumed to react with just tartaric acid
Unity g/L tartaric acid equivalents.
14
Q
Why do we degas the wine sample when measuring TA?
A
To remove CO2 because CO2 can react with H2) to form Carbonic acid (H2CO3).
This will contribute to the TA reading. We want TA due to dissolved organic acids only.
15
Q
How do we degas a wine sample when measuring TA?
A
Degas sample (to remove CO2) by boiling, placing under vacuum, or by sparging with an inert gas like nitrogen or argon.
16
Q
How do we calculate TA?
A
Titratable acidity = titration volume in mL x 0.75 g/L Tartaric acid equivalents
17
Q
How do we calculate total SO2 in a wine sample?
A
Total SO2 = free SO2 + bound SO2
18
Q
When we measure free sulfur dioxide in wine what are we measuring?
A
Three different forms of sulfur dioxide:
- Molecular sulfur dioxide
- Hydrogen sulfite
- Sulfite
19
Q
What is the distribution of forms of sulfur dioxide at wine pH?
A
- Hydrogen sulfite is the most abundant form
- Molecular sulfur dioxide concentration is very dependant on wine pH.
- Sulfite is the least abundant form at wine pH.
20
Q
Why is molecular sulfur dioxide (SO2) concentration important in wine?
A
i) active against microbes
ii) Only volatile form
iii) aroma at high concentrations
21
Q
What form of sulfur dioxide is volatile?
A
Molecular sulfur dioxide (SO2)
22
Q
Why do we add phosphoric acid to our wine sample when completing free sulfur dioxide by aspiration / oxidation?
A
- Molecular Molecular sulfur dioxide (SO2) is the volatile form
- We add phosphoric acid to sufficiently lower pH of our wine to push all the forms of free Sulfur dioxide into the molecular SO2 form
Volatile SO2 swept from wine by aspiration
23
Q
What happens to the SO2 in our wine sample when it is aspirated and passes through the condensor?
A
SO2 trapped by hydrogen peroxide (H2O2) in the pear shape flask
Hydrogen peroxide oxidises SO2 to non-volatile sulfuric acid (H2SO4)
The more SO2 aspirated from the wine the more H+ generated in the pear shaped flask
H+ changes indicator in pear shaped flask from green to purple indicating acidic conditions.
24
Q
How do we quantify the H+ present in pear shaped flask after aspiration for 10-15 minutes?
A
H+ present in pear shaped flask quantified by titration with 0.010 mol/L OH- (0.010 mol/L NaOH)
Once H+ has been consumed by OH-, indicator changes colour from purple to green signifying end point.
25
How do we calculate free SO2 concentration once we know the end point of our titration?
If the NaOH concentration is exactly 0.010 M then we use the calculation: [Free SO2] = 16 x V (ppm)
V is the volume of sodium hydroxide used in the titration in units mL
Otherse
[Free SO2] = V x C x 1.6 x 1000 (ppm)
26
When determining free SO2 by aspiration method:
The more free SO2 in the wine means ....
... more volatile molecular SO2 generated after acid addition to wine, which means ...
... more sulfuric acid that will be generated in the hydrogen peroxide solution, which means ...
... more sodium hydroxie to be used in our titration
27
How do you determine bound S2 by aspiration / oxidation?
- Repeat procedure while heating sample flask
- Heating releases sulfur dioxide from certain wine components
- Calculate bound levels using ; [Bound SO2] = 16 x V (ppm), where V = titration volume (mL)
28
Why is the condenser critical when determining bound SO2?
Condenser critical to stop volatile acids reaching pear shaped flask and stops us from distilling all wine into pear shaped flask.
29
What principle does ebulliometry use to measure the (v/v) concentration of ethanol in a sample?
Ethanol depresses the boiling point of a solution compared to water
As we increase the ethanol concentration, the boiling point of the solution will drop.
30
How do we overcome the fact that changes in atmospheric pressure also influence boiling point of a solution?
This problem overcome by measuring boiling point of water around the same time.
31
Why do we use cold water in the condenser of an ebulliometry?
Condenser required when determining the boiling point of our wine because we don't want ethanol to escape the apparatus before it boils.
32
How do we calculate the (v/v) concentration of sample using the data obtained from ebulliometry?
Sliding scale used to convert difference in boiling points (i.e., water vs wine) to ethanol concentration in wine.
33
What is the rebelein method used for?
Measuring glucose and fructose concentrations in wine
34
Why are glucose and fructose reducing sugars?
- Have open chain form that can be oxidised and in the process can reduce something else.
35
Summarise the rebelein method for reducing sugars
- Thiosulfate determines amount of iodine
- Iodine determines amount of CU2+ that didn't react with sugars (i.e., determined copper)
- Initial copper - determined copper = consumer copper
- Consumed copper allows a calculation of sugar concentration
36
How do we calculate reducing sugar concentration once we have completed the steps in the rebelein method?
Reducing sugar concentration = B - V
Where B = blank titration volume (mL)
V = sample titration volume (mL)
Units: g/L glucose equivalents
37
Summarise the ripper analysis of sulfur dioxide
Sulfur dioxide is a reducing agent - provides electrons to anything that will take them.
Iodine is a good at accepting electrons.
Once sulfur dioxide is depleted, iodine accumulates and interacts with starch to generate blue / black colour - end point.
38
Why do we standardise (determine exact concentratrion) iodine when using the ripper analysis of sulfur dioxide?
Iodine is volatile and makes it difficult to prepare accurate standards
39
Why do we add NaOH to measure total SO2 in the ripper analysis of sulfur dioxide?
Total sulfur dioxide requires binding agents to be liberated by hydrolysis - 1.2M NaOH.
This raises the pH and there are lots of OH- in the wine and they substitute onto the binding agent liberating bound SO2.
Acid is then added to remove base (acid conditions needed for titrations).
40
Summarise the procedure for ripper analysis of sulfur dioxide
1. Standardise iodine by titratrion againt thiosulfate
2. Measure free sulfur dioxide with iodine titratrion
3. Measure total sulfur dioxide wit iodine titratrion
41
What are the limitations of the ripper analysis of sulfur dioxide?
What type of wine is it best suited for?
Considered less accurate method, than aspiration / oxidation method as other wine components can be oxidised by the iodine:
- Ascorbic acid and some phenolic compounds
These interfering wine components can be accounted by binding SO2 to acetaldehyde and titrating wine by limited accuracy by this approach
Best results in white wines without added ascorbic acid
Colour of red wine and high concentratrion of phenolic compounds is problematic.
42
What happens at the end point of titratrion when conducting DCPIP analysis of ascorbic acid?
If we have wine with ascorbic acid present it reacts with the DCPIPH (light pink) to produce a colourless form.
When all of the ascorbic acid has gone there is nothing to reduce the DCPIPH and its colour will remain in solution (rose pink).
43
Summarise the DCPIP analysis of ascorbic acid
Ascorbic analyis reduced DCPIPH to a colourless form
Ascorbic acid is oxidised in the process
When no further ascorbic acid is present the DCPIPH accumulates (rose-pink colour - acidic medium)
44
Why is metaphosphoric acid reagent used instead of orthophosphoric acid in the DCPIP analysis of ascorbic acid?
- Increases stability of ascorbic acid (both the pH and structure of metaphosphoric acid contribute to ascorbic acid stability)
- Orthophosphoric acid does not protect ascorbic acid as well as metaphosphoric acid
- Metaphosphoric acid has limited stability
45
What are the advantages of the DCPIP analysis of ascorbic acid?
- Accuracy - DCPIP is selective for ascorbic acid
- SO2 influence must be removed by addition of acetaldyde during analysis
46
What are the disadvantages of DCPIP analysis of ascorbic acid?
- Frequent preparation of DCPIP and metaphosphoric acid
- Careful storage of DCPIP and metaphosphoric acid
- Still require a step to remove SO2 interference
- Require ascorbic acid standard
47
Summarise the determination of volatile acidity by Markham Still
We use a Markham still which is designed to pass steam through a wine sample, that steam carries with it volatile acids, that pass through condense and are captured in a receival flask.
Once volatile acids are seperated we need to determine amount collected by titratrion with a base.
End point determined with phenolphthalein indicator.
48
What is the main component of VA?
Volatile acid (VA) measure is reported in what units?
Acetic acid
'g/L acetic acid equivalents'
49
What is the main benefit of using a Markham still over enzymatic analysis of VA?
Markham still measures all volatile acids instead of just acetic acid and it is all of those acids that can contribute to the VA of wine.
50
What are the interfering agents when determining volatile acidity by Markham Still?
1. Sorbic acid interferes significantly with volatile acidity measure even though in wine conditions sorbic acid does not usually contribute significant aroma. Sorbic acid will significantly increase the measure.
2. Sulfur dioxide can steam distil and contribute to acidity of receiver flask. Producing hydrogen ions that can be measured. We add hydrogen peroxide to the wine to remove SO2 to produce products that do not steam distil.
3. Absorbed CO2 can contribute to acidity - titrate distillate soon after collection to avoid adsorption of carbon dioxide.
51
In addition to acetic acid, the main component of volatile acidity, what are some weaker organic acids that contribute to volatile acidity?
Valeric acid
Formic acid
Butyric acid
Propionic acid
52
When performing the 2,6-DCPIP ascorbic acid analysis, why is a standard solution of ascorbic acid prepared?
To compensate for any decomposition in the 2,6-DCPIP
53
During enzymatic analysis of glucose and fructose, which of the following is true of hexokinase? It:
Converts both glucose and fructose to their 6-phosphates.
54
The units of titratable acidity are given as “tartaric acid equivalents” because:
titration cannot distinguish the various acids in the sample.
55
Corrected hydrometer result (Bé) =
Hydrometer reading (in Bé) + [0.05 x (Measured Temp - Calibrated Temp)]
56
In the aspiration-oxidation method of sulfur dioxide analysis, a student failed to turn on the water supply to the condenser. This could result in which of the following?
Abnormally high results that are not explained just by release of bound sulfur dioxide
57
Why do the spots in the TLC experiment show up as yellow dots?
Because yellow is the acid colour of the indicator solution.
58
Enzymatic analysis of malic acid requires two enzymes. Why is this?
One enzyme oxidises malic acid and the second enzyme is needed to overcome the reversibility of the oxidation
59
How many of the following are true of the Rebelein method?
* Titration volumes of zero can occur with some wine samples.
* The quantity of starch added is critical.
* Both glucose and fructose react, but sucrose does not.
* Sodium thiosulfate is used as titrant.
* A blank titration of "0 mL" is required.
Three:
- Both glucose and fructose react, but sucrose does not.
- Sodium thiosulfate is used as titrant.
- A blank titration of "0 mL" is required.
60
What are three procedures carried out during the measurement of a wine’s VOLATILE ACIDITY that help minimise possible sources of error?
- Washing markham still
- Performing blank
- Add H2O2 to oxidise SO2
- Degassing distillate
- Adding NaOH to water in collection flask until just basic
61
A student used an ebulliometer to determine the alcohol content of a wine sample. The result obtained was several percent lower than the alcohol content shown on the bottle’s label. Assuming the label had the correct value, name and briefly describe 2 possible sources of error which caused this falsely low result?
- Not rinsing ebulliometer with wine
- No water in condenser
- Not determining boiling point on the same day
62
Why is 25% phosphoric acid added to the wine sample in sulfur dioxide analysis? Your answer must include appropriate chemical equation/s.
Sulfur dioxide can also exist in two non-gaseous forms, called bisulphite ion and sulphite ion, at wine pH.
These non-gaseous forms are not removed by the air stream.
To overcome this difficulty, the sample is strongly acidified by addition of 10 mL of phosphoric acid solution.
Under the strongly acidic conditions that are produced, the non-gaseous forms are effectively completely converted to dissolved gaseous sulfur dioxide.
Phosphoric acid provides H+ to drive equilibria to the left.
Only volatile molecular form of SO2 can be aspirated.
63
In the Rebelein method for reducing sugars, the final reaction is a titration between sodium thiosulfate and iodine.
Explain how the iodine is produced during the Rebelein procedure.
The excess of copper (II) reagent reacts with sugars to produce copper (i)
Left over copper (II) reacts with iodide to generate iodine.
64
Which of the following is correct regarding hydrometry and refractometry?
A. Both are typically utilised for the determination of ethanol concentration in wine
B. Refractometry is a direct measure of density of juice
C. Hydrometry and refractometry are only influenced by temperature once the temperature departs from 4C
D. The refractometer cannot be used for red wine due to the presence of pigments
E. None of the above are correct.
E. None of the above are correct.
65
A ferment has just finished and the pH and TA is about to be measured. Which of the following is correct?
A. The dissolved carbon dioxide will have little impact on pH but a significant impact on TA
B. The dissolved carbon dioxide will have little impact on TA but a significant impact on pH
C. The dissolved carbon dioxide will have little impact on both TA and pH
D. The dissolved carbon dioxide will have a significant impact on both TA and pH.
E. It is not possible to tell without measuring atmospheric pressue.
A. The dissolved carbon dioxide will have little impact on pH but a significant impact on TA
66
In the aspiration-oxidation method of free sulfur dioxide analysis, how many of the following would be expected to cause a large error (i.e. > 20%) in the determined free sulfur dioxide level of a white wine?
* Addition of 20mL wine and 10mL phosphoric acid to the round bottom flask that also contained approx. 1mL of deionised water
* Heating of the wine with a busen burner
* No water flowing through the condenser tubing
* 4 drops instead of 3 drops of indicator added to the hydrogen peroxide solution
* White wine of pH 3.2 instead of pH 3.5
* Addition of 10mL of wine instead of 20mL of wine
Two:
- Heating of the wine with a busen burner
- Addition of 10mL of wine instead of 20mL of wine
67
Which of the following could be a reasonable explanation for an ebulliometer whose thermometer shows an ever increasing temperature (i.e. no constant temperature below 98-100C) during the determination of the boiling point for a white wine sample with 12.0 %(v/v) ethanol?
A. The wine has too many phenolic compounds
B. The water in the ebulliometer condenser is not present or is too hot
C. The atmospheric pressure has changed and impacted the boiling point of the wine
D. The white wine sample was not sufficiently mixed before an aliquot was taken
E. The wine is American and can only be measured in Farenheit
B. The water in the ebulliometer condenser is not present or is too hot
68
What would happen if copper(I) was added during the Rebelein determination of reducing sugars in wine instead of copper(II):
A. The measured sugar in the wine would be much larger than that actually present because the added copper is already in the reduced form
B. There would be no impact on the final result (i.e. addition of either copper(I) and copper(II) would achieve the same result) as the copper ions are added in excess.
C. The blank and sample titration values would be both equal to 0 mL
D. Only glucose and not fructose would be measured
E. Addition of copper(I) to a water solution would result in a violent explosion
C. The blank and sample titration values would be both equal to 0 mL
69
What is the main purpose of the standard ascorbic acid solution prepared during the determination of ascorbic acid in wine by the DCPIP method?
A. To fill in time before lunch
B. To ensure the purity of the solid ascorbic acid utilised for the method
C. To avoid inaccuracy associated with the volatility of ascorbic acid solutions
D. To neutralise and sulfur dioxide that may interfere in the determination
E. To allow quantification of the titration value of the sample and also to account for any deterioration in the DCPIP reagent over time
E. To allow quantification of the titration value of the sample and also to account for any deterioration in the DCPIP reagent over time
70
During the titration performed in the measurement of ‘Volatile Acidity’ by the Markham still, what is actually occurring:
A. the volatile acids that are in the wine are donating protons to hydroxide ions that are being added via a Markham still burette directly to the wine.
B. the volatile acids that have been steam distilled from the wine are donating protons to hydroxide ions that are being added via a burette.
C. the sodium hydroxide that has been steam distilled from the wine are accepting protons from the volatile acid that is being added via the burette in the form of acetic acid.
D. Acidic compounds are steam distilled from wine and then become volatile in alkaline solution once sodium hydroxide is added to the distillate.
E. Hydroxide ions are consumed until the end point of pH 8.2 is reached (or pH 7.0 in the USA).
B. the volatile acids that have been steam distilled from the wine are donating protons to hydroxide ions that are being added via a burette.
71
What is the most typical reason for performing thin layer chromatography during wine production:
A. To determine the main volatile acid present in white wine
B. To assess the acid concentrations responsible for a given TA
C. To assess if malolactic fermentation is near completion
D. Because someone has smashed all the aspiration-oxidation apparatus
E. To assess the acid strengths of the respective acids in the wines (i.e. pKa values)
C. To assess if malolactic fermentation is near completion
72
Which agent below is most responsible for the high selectivity in determining malic acid concentration in wine by the enzymatic analysis (i.e. high selectivity = the methods ability to be specific for malic acid):
A. The suite of enzymes utilised
B. The use of accurate and calibrated micropipettes
C. The use of a spectrophotometer at 340 nm rather than 365 nm
D. None – it has poor selectivity in the presence of tartaric acid
E. An accurate malic acid standard solution that is provided
A. The suite of enzymes utilised
73
What is the most appropriate medium to demonstrate the formation of acetic acid by wine spoilage bacteria?
A. MRS + apple juice agar
B. WLN agar
C. lysine agar
D. GYC
D. GYC
74
Which of the following produces hat-shaped ascospores?
A. Saccharomyces cerevisiae
B. Pichia anomala
C. Schizosacharomyces octosporus
D. Debaryomyces hansenii
B. Pichia anomala
75
Saccharomyces cerevisiae is generally characterised by which shape?
A. spheroidal
B. flask
C. elongated oval
D. apiculate
A. spheroidal
76
Cycloheximide (actidione) is important in the wine microbiology diagnostic laboratory. What does cycloheximide do? (1 mark). Give two examples of cycloheximide use in a wine laboratory (2 marks).
Cycloheximide inhibits eukaryotic cell division, so it will inhibit the growth of yeasts. It is used to inhibit the growth of yeasts when you wish to enumerate the number of lactic acid bacteria. Similarly it can be used for estimating spoilage bacteria in a ferment.
77
You decide to use a haemocytometer to count the number of yeast cells in a ferment. Describe the procedure you would use to accurately use the Neubauer haemocytometer to perform your cell count. (6 marks)
Dilute the cell suspension so that the number of cells visible down the microscope can be counted.
Ensure the coverslip makes good contact with the haemocytometer
Do not count cells in squares where air bubbles or debris are present
Count at least five squares and perform at least three determinations
Use a convention that includes cells touching the left or top margins and excludes cells touching the bottom and right margins
Include the budding cells and express these as a proportion of the total number of cells.
78
Two different methods have been utilized to measure bound sulfur dioxide in wine during the laboratory classes. Name one method and explain how the bound sulfur dioxide is converted to free sulfur dioxide in that particular method. (5 marks)
Aspiration-oxidation method - the acidified wine sample is heated and this releases bound sulfur dioxide into the free form.
79
Describe how the DCPIP reagent is able to measure ascorbic acid concentration. In your answer indicate i) the type of reaction between DCPIP and ascorbic acid, ii) why a colour change occurs to signify the end of the titration, and iii) indicate how selective the DCPIP reagent is for ascorbic acid (including interfering agents). (6 marks)
i) REDOX reaction OR DCPIP oxidises ascorbic acid
ii) The colour change occurs as the DCPIP reagent is able to accumulate in the acidic sample solution after ascorbic acid is exhausted.
iii) The DCPIP is very selective for ascorbic acid as long as sulfur dioxide is not present
80
How does the ebuilliometer allow the determination of alcohol concentration in wine?
A. Ethanol in water causes a depression in the boiling point of the mixture compared to pure water. The extent of boiling point depression can be related to the ethanol concentration in the mixture.
B. The ethanol is distilled from the wine and the temperature of the evaporating ethanol indicates its concentration in the wine.
C. Hydrolysis of the ethanol from compounds in the wine releases energy which is measured by the ebuilliometer. The higher the ethanol concentration, the higher the measured temperature release from the wine.
D. The heating wine allows a reaction between the sugar and the ethanol in the wine, and this leads to a characteristic boiling temperature of the wine. It can be only used in wines that contain at least some sugar.
A. Ethanol in water causes a depression in the boiling point of the mixture compared to pure water. The extent of boiling point depression can be related to the ethanol concentration in the mixture.
81
Why does iodine require standardisation with thiosulfate in the Ripper method for the determination of sulfur dioxide in wine?
A. Iodine is volatile and requires standardisation to determine its accurate concentration.
B. Iodine requires activation with thiosulfate prior to reacting with sulfur dioxide.
C. Thiosulfate is the form in which sulfur dioxide is added to wine.
D. The thiosulfate is required to unbind sulfur dioxide in wine.
A. Iodine is volatile and requires standardisation to determine its accurate concentration.
82
In the measurement of free sulfur dioxide by the aspiration-oxidation method, why is the phosphoric acid required?
A. To convert all free sulfur dioxide to a volatile form that can be swept from the wine, with aspirating air, and delivered to a separate hydrogen peroxide solution.
B. To oxidise sulfur dioxide to sulfuric acid so that it can be titrated.
C. To release sulfur dioxide from binding agents.
D. To allow measurement of free sulfur dioxide but is not required to measure bound sulfur dioxide.
A. To convert all free sulfur dioxide to a volatile form that can be swept from the wine, with aspirating air, and delivered to a separate hydrogen peroxide solution.
83
Why is the condenser critical in the determination of free sulfur dioxide by the aspiration oxidation method of analysis?
A. To avoid volatile acids reaching the solution of hydrogen peroxide.
B. It ensures that the gas impacting the hydrogen peroxide solution is sufficiently cool to allow reaction between hydrogen peroxide and sulfur dioxide.
C. It converts the free sulfur dioxide to a non-volatile form.
D. It is not critical for the determination of free sulfur dioxide.
D. It is not critical for the determination of free sulfur dioxide.
84
Which of the following are least likely to be units of titratable acidity in wine analysis?
A. g/L tartaric acid equivalents.
B. g/L sulfuric acid equivalents.
C. g/L sodium hydroxide equivalents.
D. g/L malic acid equivalents.
C. g/L sodium hydroxide equivalents.
85
Which of the following is not a feasible reason to perform thin layer chromatographic analysis of wine:
A. To determine if a Shiraz has completed malolactic fermentation.
B. To assess if a Cabernet Sauvignon has commenced malolactic fermentation.
C. To determine the titratable acidity of a Riesling.
D. To assess if a wine contains any succinic acid.
C. To determine the titratable acidity of a Riesling.
86
Which of the following is FALSE regarding determination of sugar concentration by refractometry?
A. A relatively small sample volume is required by refractometry compared to hydrometry.
B. Refractometry measures the extent that light is ‘bent’ by a solution.
C. Refractometry makes use of the fact that increased concentration of sugar in water results in a change in ‘bending’ of incident light on the solution.
D. Refractometry is able to estimate sugar concentrations in wine.
D. Refractometry is able to estimate sugar concentrations in wine.
87
Why is hydrogen peroxide necessary in the determination of volatile acidity in wine when using a Markham still?
A. It converts volatile acids into a form that enables their steam distillation and subsequent collection.
B. It releases the volatile acids from components in the wine that bind them. Therefore, it allows the determination of total volatile acid concentration in the wine.
C. It enables conversion of ethyl acetate to acetic acid.
D. It converts sulfur dioxide into a non-volatile form that cannot undergo steam distillation and subsequent interference of the volatile acidity measurement.
D. It converts sulfur dioxide into a non-volatile form that cannot undergo steam distillation and subsequent interference of the volatile acidity measurement.
88
What is responsible for the end point colour change in the determination of ascorbic acid in wine by DCPIP:
A. Ascorbic acid reduces the DCPIP during the titration to form a rose/pink coloured product.
B. The absence of ascorbic acid at the end point of the titration allows the DCPIP to accumulate and in the acidic titration solution it adopts a pink/rose colour.
C. The ascorbic acid is oxidised by DCPIP, and at the end point of the titration the oxidation products of ascorbic acid interact with starch to form a dark coloured end point.
D. There is no colour change as the end point is determined by a pH meter.
B. The absence of ascorbic acid at the end point of the titration allows the DCPIP to accumulate and in the acidic titration solution it adopts a pink/rose colour.
89
Which of the following statements is NOT true about Schizosaccharomyces octosporus?
A. During sexual reproduction produces ascospores
B. Can be enumerated using a haemocytometer
C. Divides by budding
D. Is larger than Lactobacillus
C. Divides by budding
90
12. Which of the following is most likely to have a pellicle when grown in a broth culture?
A. Pichia anomala
B. Saccharomyces cerevisiae
C. Schizosaccharomyces octosporus
D. None of these yeasts produce a pellicle in broth culture
A. Pichia anomala
91
The following procedure was carried out to check the sterility of wine pre-bottling. A 50 mL wine sample was filtered and the filter paper placed on to a WL media agar plate. After appropriate incubation three individual yeast colonies had grown. Which of the following most accurately conclusion from these findings?
A. The wine is likely to be cloudy
B. Acetic acid (vinegar) aroma is likely to be present
C. The wine contains some non-Saccharomyces contaminating yeasts
D. The wine is likely to be clear
D. The wine is likely to be clear
92
What is wrong with the following statement?
Saccharomyces cerevisiae is Gram Positive.
S. cerevisiae is a yeast, it is neither gram positive nor gram negative. The gram stain is used to differentiate different types of bacteria, not yeasts.
93
What do the ‘G’ ‘Y’ and ‘C’ stand for in GYC agar?
Glucose
Yeast (Extract)
Calcium carbonate
94
What is GYC used for in the wine microbiology laboratory?
Detection of acetic acid bacteria
95
How does GYC agar work?
Acetic acid produced by acetic acid bacteria dissolves the insoluble calcium carbonate leading to a clear zone around the growth of the bacteria.
96
You wish to enumerate the number of yeast cells in a ferment using a serial dilution platting method.
You remove a 1 mL aliquot of the ferment and serially dilute it. You then plate out 0.1 mL volumes of the 10-4,10-5 and 10-6 dilutions to MEA plates. After a suitable incubation period you examine the plates. The diagrams below indicate what you observe.
The 10-4 plate has too many and the 10-6 plate has too few colonies to be accurate. There are 36 colonies on the 10-5 plate.
As only 0.1 mL was platted out, the 10-5 dilution has 360 CFU per mL.
Therefore the CFU / mL in the undiluted ferment is 3.6 x 107 per mL.
97
Discuss the advantages and disadvantages of serial dilution of the yeast culture method compared to the haemocytometer for enumerating microorganisms.
Advantages of the plate method are that only viable cells grow, whereas in the haemocytometer you don’t know if the cells are alive or dead. (Although you could use a differential stain to detect viability if
you wanted to).
Disadvantages of the plate method are that it takes time for the cultures to grow, you don’t know if a colony has arisen from one cell or a clump of cells. Another disadvantage is that not all microorganisms will grow on the media, i.e. you need different media and different culture conditions (e.g. aerobic vs
anaerobic) to culture different microorganisms.
An advantage of the haemocytometer is that you can do a number of cell counts relatively quickly. You can also assess the degree of budding with the haemocytometer. You do not know however, if the nonbudding cells are alive or not.
98
Describe the manner by which the Markham Still enables the determination of volatile acidity in wine?
Steam passed through sample bringing volatile acids with it to a condenser
Volatile acids condense in condenser and are collected in a distillate container.
Distillate is titrated with 0.05 M sodium hydroxide to determine acid concentration.
99
Describe how bound sulfur dioxide is released from binding agents in the aspiration-oxidation method, and also how it is released in the Ripper method?
Heat, combined with acidic conditions and aspiration, is ued to release SO2 from binding agents in A-O method.
Alkaline conditions are used to release bound SO2 in the ripper method.
100
What factors impact cell shape of yeasts?
- Age
- Physiological conditions
- Asexual reproduction
101
Name four types of yeast budding:
- Multilateral
- Bipolar
- Unipolar
- Multiple bud scars
102
What is one yeast common in the production of wine that undergoes binary fission?
- Schizosaccharomyces
- Cross wall formation between cells
- No or little cell wall constriction
103
What is one yeast common in the production of wine that is part of the division ascomycota?
- Schizosaccharomyces
or
Pichia anomala
ascomycota > Ascospores
104
What is one yeast common in the production of wine that forms hat shape ascospores?
Pichia anomala
105
A student is preparing to plate out S. cerivisiae. Why should they not choose a Lysine agar? What medium would be suitable for S. cerivisiae?
S. cerivisiae can not utilise lysine as a source of Nitrogen
Media containing 12% ethanol alcohol and some metabisulfite
106
What type of media is used for yeast estimatation?
Malt extract, grape juice agar and WL agar for total yeasts.
107
Why is serial dilution used in the isolation and enumeration of yeasts?
Serial dilution allows us to back count number of colonies in an original sample containing too many CFU to count.
108
What type of media is best used for the isolation and enumeration of LAB?
- MRS + Apple of Tomato juice (20-25%) + 2-3% agar
- pH 4.7 for fastest growth
- Preger anaerobic environments
- cycloheximide limits growth of yeasts
109
What type of media is best used for the isolation and enumeration of Acetic Acid Bacteria?
- GYC (Glucose, Yeast Extract, Calcium Carbonate) with cycloheximide
110
What are two main species of AAB important to wine production?
- G. oxydans
- A. hansenii
111
What principle does the Ripper method utilise to measure sulfur dioxide?
- SO2 acts as a reducing agent - it donates electrons
- Utilises ability of SO2 to reduce iodine which readily accepts electrons.
- Reaction much more efficient in acidic conditions so conducted in the presence of sulfuric acid
- Iodine concentratrion standardised by titration with sodium thiosulfate
112
What reaction occurs between ascorbic acid and 2,6-DCPIP?
What effect does SO2 have on the reaction?
- Ascorbic acid rapidly reduces DCPIP from blue to almost colourless form
- SO2 can react with DCPIP and contribute some inaccuracy so it must be tightly bound to a carbonyl compound during analysis
113
What is the purpose of the GOT enzyme in the determination of malic acid concentration by enzymatic analysis?
Reverse reaction is more favourable so GOT is added to pull the reaction to the right.
114
The Rebelein method uses a back titration. Explain how?
- Known excess of reactant is added
- All reagent (e.g., sugar) is consumed and some reactant is left over (Cu (II))
- Amount left dependent on amount of reagent present initially
- Excess reactant determined by titration
- Quantity of reagent indicated by difference between titration value and titration value when no reagent is present
115
What is the most complete TA unit?
g/L tartaric acid equivalents (at pH 8.2)
116
What would have the highest TA?
- Wine sparged with Nitrogen
- Wine sparged with CO2
- Wine from recently finished ferment
TA impacted by CO2 so TA highest when sample sparged with CO2.
TA will also be impacted if sample taken from wine that has just finished fermenting and is saturated with dissolved CO2.
117
If it a specific hydrometer doesn't sink in a juice sample, what does it mean?
The specific hydrometer is suited to a solution with less sugar.
118
Why is molecular SO2 volatile?
Why are other forms non-volatile?
SO2 has no charge so is volatile.
The following are non-volatile forms because they have a charge and can't escape solution or are bound to a carbonyl:
Hydrogen sulfite HSO3-
Sulfite SO32-
Bisulfite addition product of acetaldehyde
119
What are the indicator colours and end-points used in the A-O method?
- Purple - acidic
- Green - alkaline
120
What causes colour change at the end-point of the titration for the ripper method?
The absence of free SO2 which allows iodine to accumulate and interact with the starch forming a grey/ black colour.
121
Why do we call glucose and fructose reducing sugars?
Reducing sugars can reduce Cu (II) to Cu (I)
Reduction is gain. Therefore, Cu+ is formed from Cu2+ by the loss of one electron.
122
Why does is blank titration in the analysis of reducing sugars, typically lower than the wine titration?
During the Rebelein method, the blank titration typically provides a higher value than the wine because there is more iodine present in the blank solution than in the wine solution. This is because sugars in the wine lead to decreased iodine production.
As the concentration of reducing sugar increases, and the amount
of excess copper reagent decreases, less copper (II) reagent is available to produce iodine from iodide ion.
123
What would have a higher Rf value? Tartaric or acetic acid.
Acetic acid because it's a weaker acid than tartaric acid.
TCC analysis means it would travel further and therefore would have a higher Rf value compared to tartaric acid.
124
What are the correct units for determination of VA by Markham still?
g/L acetic acid equivalents
125
What happens if the condenser is not used when determining VA with a Markham still?
If it is not used the 150 mL of distilate would probably never be collected as the steam passing from the wine sample would not condense and would escape as steam.
126
True or false?
Vast majority of white wines require dilution prior to enzymatic analysis for malic acid
True
127
True or false?
Enzymatic analysis of malic acid in white wine utilises a single type of enzyme in the analysis
False
128
True or false?
The precision of enzymatic analysis of glucose can be determined by the comparison of the determined result to a standard
False
This is measuring accuracy rather than precision
Precision is how close repeat measurements are to each other.
