The impact of yeast strains on the production of thiol-type aromas in Bacchus
Christine Gentry and Rory Loftus
Introduction
The trial sought to assess the impact upon wine made from Bacchus grapes of fermentation with
different strains of Saccharomyces cerevisiae; in particular, the extent to which yeast affects wine
aroma. It has previously been demonstrated that grape varietal aromas can be significantly affected
by yeast strain selection (Howells, et al, 2004).
Volatile Thiols
The substances responsible for the varietal aromas of wines are volatile thiols, sulphur-containing
compounds produced from odourless precursors in grape must by the action of yeast during alcoholic
fermentation (Roland, et al, 2011). These thiol precursors include cysteine conjugates and glutathione
precursors (Robinson, et al, 2014). The enzyme activity which occurs in liberating volatile thiols from
their precursors varies between yeast strains (Murat, et al., 2001).
Table 1 shows the thiols commonly expressed in varietal white wines, including Bacchus.
Table 1: Key volatile thiols identified in white wine aroma and their structure.
Volatile thiol
Structure (Dubourdieu, et al, 2006)
4MMP
4-mercapto-4-methylpentan-2-one
CH3
I
CH3 – C – CH2 – C – CH3
I
II
SH
O
3MHA
3-mercaptohexyl acetate
CH3 – CH2 – CH2 – CH – CH2 – CH2 – O – C – CH3
I
II
SH
O
3MH
3-mercaptohexan-1-ol
CH3 – CH2 – CH2 – CH – CH2 – CH2 – CH2 – OH
I
SH
Table 2 shows the aromas commonly associated with these thiols, and their perception thresholds.
These are low as sulphur compounds have a high impact on aroma, the sulphur group binding strongly
to the nose’s olfactory receptors (Fisher & Scott, 1997).
1
Table 2: Key volatile thiols responsible for varietal aroma in wine, aromas associated with them (Swiegers
et al, 2005), and their perception thresholds in a dilute alcohol solution (Ribereau-Gayon, et al., 2006).
Volatile Thiol
Aromas
Perception
threshold (ng/L)
4-mercapto-4-methylpentan-2-one
4MMP
Box tree, broom, blackcurrant bud
Cat urine at high concentrations
0.8
3-mercaptohexyl acetate
3MHA
Passion fruit, box tree
4
3-mercaptohexan-1-ol
3MH
Passion fruit, grapefruit, citrus
60
It has previously been demonstrated in research on Sauvignon blanc and other varieties that yeast
strains can be selected to enhance thiol-type aromas associated with 4MMP, 3MH and 3MHA. This
trial sought to ascertain if this was the case in Bacchus.
4MMP has been identified as being present in Bacchus (Schneider, et al., 2003), having long been
proposed as the source of blackcurrant aroma (Rapp & Pretorius, 1990).
3MH is the most abundant thiol in wine, and undergoes esterification with acetic acid to form 3MHA
(Coetzee, et al, 2012). Yeast strains have been shown to impact upon the conversion of 3MHA from
3MH (Swiegers, et al., 2005), changing aromas released.
3MH has not been studied in Bacchus, although 3MH and 3MHA have been identified in Riesling and
Sylvaner (Tominaga, et al., 2000), both varieties closely related to Bacchus.
Method
Micro-vinifications with five treatments were carried out using four yeasts intended to express thioltype aromas and a control yeast, details of which are shown in Table 3.
Processing
Bacchus grapes from Plumpton College’s Rock Lodge vineyard were whole-bunch pressed and cold
settled at 10°C. Additions of Sulphur Dioxide (SO2)) as PMS at 60mg/L, 40mg/L ascorbic acid, and
80mg/L Extralyase were made. Potential alcohol was assessed by hydrometry at 10.3%.
Turbidity was assessed by turbidity meter at 28.4 NTU, and increased to 107 by the addition of 2L of
lees, bringing it within the recommended range of 100-250 NTU (Wilker, 2010).
Titratable acidity (TA), pH and free SO2 were measured, and PMS added to bring free SO2 levels to
20mg/L, within the recommended range at pH 3.17 (Rankine, 2004).
2
Table 3: Yeasts used in the trial, showing fermentation characteristics summarised from manufacturers’
product data sheets. (Institut Œnologique de Champagne, 2014a) (Institut Œnologique de Champagne,
2014b) (Laffort, 2013a) (Laffort, 2013b) (Laffort, 2014)
Yeast
Ferment
temp. (°C)
Alcohol
tolerance
(volume)
Nitrogen
required
Turbidity
(NTU)
Aromatic characteristics (white wines)
IOC 18-2007
8-30
>15%
Not stated
None stated
IOC
Révélation
Thiols
15-25
15%
Not
stated
Low.
Use
complex
nutrients
20-80
Liberates 3MH thiol, contributing citrus
and passion fruit aromas. Lesser
accentuation of 4MMP, limiting vegetable
notes.
Zymaflore X5
13 +
16%
Medium
to high
<50
Reveals thiol-type varietal aromas - 3MH,
3MHA, especially 4MMP, citrus, tropical
fruits and boxwood. Fruity and floral
fermentation aromas
Zymaflore
VL3
15-21
14.5%
High
Not stated
(example
is 40
NTU)
Reveals thiol-type varietal aromas 4MMP, 3MH, 3MHA; boxwood, broom,
citrus, passion fruit. Improved mouthfeel,
suitable for ageing.
Zymaflore
Delta
14-22
15%
High
>150
Enhances varietal aroma (grapefruit,
passion fruit, mango, and lychee). High
capacity to enhance 3MH & 3MHA, lower
capacity to express 4MMP (tomato leaf,
boxwood).
16-18 is
ideal
Inoculation
The juice was divided into 10 demijohns and inoculations with each of the five yeasts carried out in
duplicate. Yeast was added at the rate of 20g/hl, rehydrated in ten times its weight in water at 38°C
and left for 20 minutes before addition to must.
Fermentation
After inoculation, fermentation conditions were kept as near identical as possible to minimise
extraneous variables (Northledge, et al., 1997). Individual trials received the same additions, and
were fermented at near-identical temperatures using climate control cabinets, with density and
temperature monitored on a daily basis. Organoleptic sampling was performed daily to taste for
oxidation or Hydrogen Sulphide (H2S).
Fermentation temperature was 13-15°C until day 10. By this point ferment was almost complete and
to avoid stuck ferments, fermentation was completed at 21-22°C; by day 14 all ferments had finished.
Alcohol was assessed by ebulliometry, PMS added at 50mg/L and the trials moved to the 5°C cabinet
for cold settling, before being blended. Wines were tested for pH and SO2, and SO2 levels were
increased to 20mg/L by PMS addition before bottling.
Fining was not carried out, as bentonite fining can bind aromatic compounds, reducing wine aroma
(Butzke, 2010), which would inhibit sensory evaluation.
3
Sensory Evaluation
A summary of the trial was presented to a panel of 15 assessors (Plumpton wine students), identifying
key volatile thiols responsible for varietal aromas and the aromas associated with them (Table 2).
A blind tasting was conducted using the Plumpton tasting room. To minimise sequence error, where
perception is distorted by order of presentation, the order of assessment for each taster was
determined by a Williams Latin Square (Jackson, 2002)
The panel were asked to consider the extent to which each wine displayed the aromas associated
with volatile thiols 4MMP, 3MH and 3MHA, and to: a) Rank the wines according to thiol intensity;
b) List the aromas detectable from each wine (not including faults);
c) List the flavours detectable in each wine;
d) Provide any other comments on the wine (including, if relevant, any faults).
Results
Summary
Prior to inoculation, the characteristics of the juice were as shown in Table 4.
Table 4: Table showing characteristics of the juice at time of inoculation.
Density
pH
Free SO2
(mg/L)
Turbidity
(NTU)
Titratable
acidity (mg/L)
Estimated potential
alcohol (%)
1.078
3.10
12.8
110
8.025
10.3
Post fermentation and immediately prior to bottling, the characteristics of the individual trial wines
were as shown in Table 5.
Table 5: Table showing characteristics of the trial wines post-fermentation prior to bottling,
showing pH, Free SO2 and ABV assessed by ebulliometry.
Trial wine
pH
Free SO2 (mg/L)
ABV (%)
18-2007
3.17
15
10.5
Révélation Thiols
3.18
16
10.5
X5
3.20
14
10.4
VL3
3.04
14
10.4
Delta
3.15
16
10.5
4
Fermentation
All of the duplicate ferments, bar the two X5 ferments, were near identical in their rate of fermentation
as shown in fermentation graph Figure 6. The graph shows Delta to have been an extremely fast
ferment, with VL3 and X5 also rapid. X5#1 was sluggish, but completed after ferment temperatures
were increased.
Delta, VL3 and X5 all experienced issues with H2S which were resolved by aeration, and are
discussed below.
1.075
1.065
Specific Gravity
1.055
1.045
1.035
1.025
1.015
1.005
0.995
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Day of ferment (day 0 is pre-inoculation, day 1 is the evening of inoculation)
18-2007
Rev Thiols
X5 1
X5 2
VL3
Delta
Figure 6: Fermentation graph for micro-vinifications, showing mean ferment rates for 18-2007, Rev Thiols, VL3
and Delta, and both X5 ferments. Days 1-10 were conducted at 13-14°C, days 11-14 at 21-22°C
Sensory data
The sensory evaluation of the data resulted in each wine being assigned a rank by each taster for the
intensity of its thiol-type aromas. The data obtained was checked for outlying values, and one outlier
removed (Kemp, et al, 2009), where an assessor attributed the same ranking to multiple samples
The significance of the results was evaluated using the rank sums (statistical calculations are below).
Table 7 shows which samples are significantly different to each other with a 5% significant level.
5
Table 7: Summary of ranking results for trial wines (1 low to 5 high),
showing median ranks, rank sum and significance (to 5%). Samples
sharing the same letter are not significantly different from each other.
Trial wine
Median rank
Rank sum
Significance
Révélation Thiols
4.5
55
A
VL3
4
52
A
X5
3
41
AB
Delta
2
31
B
18-2007
2
31
B
Révélation Thiols and VL3 displayed significantly more pronounced thiol-type aromas than 18-2007
and Delta.
X5, 18-2007 and Delta are not significantly different from each other, and X5 is not significantly
different from Révélation Thiols and VL3.
Statistical calculations
Significance of results - calculations for Friedman statistic (T).
The significance of the results was evaluated using the rank sums shown in Table 7 above.
The Friedman statistic (T) was calculated to demonstrate whether significant difference to 5%
existed between two or more of the different trials.
T = (12ΣR2/bt(t+1)) – (3b(t+1)), where t is the number of samples (5), b the number of assessors
(14) and R the rank sum (Kemp, et al., 2009).
The results produced a T statistic of 14.64.
As 9.49 is the critical value required to show a significant difference to 5% for 14 assessors and 5
samples (Kemp, et al., 2009), two or more of the figures can therefore be said to display a significant
difference, indicating a significant difference in the ranking of the wines.
Least significant difference - calculations for multiple comparisons of samples
To determine which sample(s) differed from the others, the least significant difference was calculated.
It can be shown that two samples are significantly different to a significance level of 5% if the
difference between the sum of their ranks exceeds the least significant difference, using the following
equation (Bewick, et al, 2004):
[Ri – Rj] > t x √2(b∑𝑘𝑗=1 ∑𝑏𝑖=1 𝑟𝑖𝑗2 )/(b-1)(k-1)
6
Where k= the number of samples, and b= the number of assessors, Rj the sum of the ranks for each
treatment, 𝑟𝑖𝑗 the rank of each individual observation.
t is the value from the t distribution for a 5% significance level, and (b-1)(k-1) degrees of freedom (52
degrees of freedom). This can be obtained from standard tables and here is 2.01.
The calculations are as follows: t x √2(b∑𝑘𝑗=1 ∑𝑏𝑖=1 𝑟𝑖𝑗2 )/(b-1)(k-1)
= 15.00
As such those pairs with a difference of more than 15 between ranks are significantly different to the
5% significance level. Table 7 above has shown which pairs differ in this way.
Aromas detected
A summary of the major aromas detected by the panel for each wine is shown at Figures 8-12
below. Only those aromas observed by more than one assessor are shown, and 1 mark was
attributed per reference, with ½ a mark attributed for qualified references such as “slight” or “hint of”.
References by assessors
7
6
5
4
3
2
1
0
Aroma
Figure 8: Major aromas detected through sensory evaluation in wine made using 18-2007 yeast.
7
References by assessors
7
6
5
4
3
2
1
0
Aroma
Figure 9: Major aromas detected through sensory evaluation in wine made using Révélation Thiols yeast.
References by assessors
7
6
5
4
3
2
1
0
Aroma
Figure 10: Major aromas detected through sensory evaluation in wine made using X5 yeast.
References by assessors
7
6
5
4
3
2
1
0
Aroma
Figure 11: Major aromas detected through sensory evaluation in wine made using VL3 yeast.
8
References by assessors
7
6
5
4
3
2
1
0
Aroma
Figure 12: Major aromas detected through sensory evaluation in wine made using Delta yeast.
Discussion
The results confirmed that fermentation with different yeasts would produce a significant difference in
the strength of thiol-type wine aromas.
The trial therefore indicates that yeast strains can have a significant effect on varietal aromas in
English Bacchus. This is of use to UK winemakers wishing to make wines displaying the varietal
aromas of the grape which is the 3rd most widely planted in the UK (English Wine Producers, 2015).
It may also be of interest to those wishing to reduce levels of certain aromas; at high levels 4MMP
can have an undesirable cat urine aroma (Swiegers, et al, 2009).
Other factors in the winery and vineyard can affect thiol precursor levels; ripeness, Botrytis infection
and skin contact all increase thiol precursors (Roland, et al., 2011). However, the concentration of
thiols released is not directly proportionate to the amount of precursor compounds in must (Pinu, et
al., 2012). Yeast is therefore an important tool for the winemaker.
The trial did not seek to identify differences in strength of individual thiols, although figures 5-9 indicate
the assessors detected aromas associated with 3MHA and 3MH (passion fruit, grapefruit, citrus);
although aromas associated with 4MMP (box tree, broom) were only apparent with Delta yeast. Table
3 shows the extent to which the yeasts claim to express these thiols.
The wines which were ranked as having strongest thiol-type aromas (Révélation Thiols, VL3) also
had the greatest agreement about the presence of an aroma, with 6-7 references to grapefruit,
associated with 3MHA / 3MH.
Yeast performance
Révélation Thiols and VL3 produced thiol characteristics, as expected, as did X5, although an
insufficient amount to show significant difference.
Delta showed some individual observations of thiol-type aromas of pink grapefruit & box tree, and
overall it produced results that were ranked similarly to that of the control yeast, 18-2007. Delta’s
performance could possibly be due to sub-optimal fermentation conditions (see below) and the need
to aerate it to minimise H2S; aeration in late-stage fermentation, which can detrimentally affect a
wine’s thiol characters (Roland, et al, 2011).
9
Limitations
Identical ferment parameters were used to minimise extraneous variables influencing the trial.
However, this resulted in some yeasts having sub-optimal ferment conditions, as shown in Table 13.
Table 13: Comparison of trial parameters with ideal fermentation conditions as shown in manufacturers’
product data sheets. (Institut Œnologique de Champagne, 2014a) (Institut Œnologique de Champagne,
2014b) (Laffort, 2013a) (Laffort, 2013b) (Laffort, 2014). Green indicates the trial conditions were within
ideal parameters for the yeast, amber indicates conditions were slightly less than optimal, and red
indicates that the trial conditions were far from ideal for the yeast.
Fermentation
temperature
Alcohol tolerance
(volume)
Nitrogen required
Turbidity (NTU)
Actual
ferment
conditions
12-22°C
(13-14°C for
majority of ferment)
10.5%
Low
(no DAP or other
nutrients added)
110
18-2007
8-30°C
>15%
Not stated.
However states “use
complex nutrients”
Not stated
Révélation
Thiols
15-25°C
15%
Low
20-80
X5
13°C+
(16-18°C ideal)
16%
Medium to high
<50
VL3
15-21°C
14.5%
High
Not stated (example
shown is 40)
Delta
14-22°C
15%
High
>150
In particular, VL3, Delta and X5 had high nitrogen requirements. Di-ammonium phosphate (DAP) was
not added, although 100-120mg/L is standard as lack of inorganic nitrogen can cause yeast to break
down grape proteins, giving rise to H2S (Boulton, et al, 1996). It is likely this contributed towards H2S
issues with those ferments.
It is possible that failure to add DAP may also have resulted in weaker varietal aromas as DAP can
influence the aroma / flavour of wines; however ammonium ions can also inhibit the thiol precursor
cysteine conjugates in varietal wines and to prevent this DAP should only be added mid ferment
(O’Kennedy & Reid, 2008).
Révélation Thiols, VL3 and Delta were fermented below optimum temperature. As 4MMP, 3MHA and
3MH thiol release is higher at 20°C than 13°C in Sauvignon blanc ferments (Masneuf-Pomarede, et
al., 2006), this may have resulted in reduced aromas.
10
Both temperature and nitrogen availability can also affect thiol release (Roland, et al, 2011). Finally
juice turbidity can also influence yeast performance and sulphide release, with the trial conducted at
a turbidity below the recommended level for Delta and above that for X5 and VL3.
To explore the effects of these limitations, future trials could be conducted to ascertain the effect on
varietal aromas of fermentation at optimal conditions for the individual yeast strains, and if the
significant difference in thiol-type aromas observed between yeasts is reduced or increased as a
result.
The authors are grateful to John Cottle for his contribution to the trial.
11
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