The Effect of Temperature on the Fermentation Rate and by fsd65350

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									     The Effect of Temperature on the Fermentation Rate and Production and
                          Retention of Yeast Volatiles

                                    Erik L. Kramer
                                 VEN 124, Spring 2001

Introduction

The important role that temperature plays in wine fermentations cannot be
overemphasized. It is clearly one of the most crucial variables to monitor during the
fermentation. Research regarding the influence of temperature on the fermentation and
wine quality has yielded a complex mixture of results.

With respect to the impact of temperature on yeast growth rates, higher temperatures
generally lead to increases in fermentation rates. Peynaud‟s first law of fermentation
asserts that fermentations proceed more rapidly at higher temperatures (Peynaud,
1984). Charoenchai et al. performed an experiment testing the effect of temperature on
the fermentation rate for 22 different strains of yeast (Charoenchai, Fleet, and
Henschke, 1998). In general, the outcome for all the yeast tested in the experiment
indicated that greater temperatures yielded increased growth rates. However, some
different species of yeast have may behave differently at different temperatures.
Ethanol tolerance and subsequent growth rates in non-Saccharomyces yeast, such as
Klockera apiculata, have been shown to improve with lower temperatures (Fleet, Heard,
and Gao, 1989). Saccharomyces cerevisae and Saccharomyces byanus have tended to
be more ethanol tolerant at higher temperatures, which explains their general
dominance in wine fermentations. Work by Fleet et. al. indicated an ethanol tole rance
of 15% at 20C for Saccharomyces cerevisae, but only 13% for Candida stellata and 7%
for Klockera apiculata (both at 20C). The general increases in yeast growth rates
correlating with higher temperatures have their limitations. Radical temperature swings,
either high or low, can lead to disruption of the plasma membrane (Boulton, Singleton,
Bisson and Kunkee, 1998). When these temperature swings are combined with high
concentrations of ethanol, the yeast cell viability is substantially compromised.

Production and retention of volatile wine components is an additional area in which
temperature plays an extremely significant role. The volatile end products produced by
yeast during the fermentation can lead to increased complexity in wines. This spectrum
of aromatics is quite broad and their presence in wine is strongly influenced by
temperature.     Desirable esters can be formed as end products of the yeast
fermentation. However, temperatures in excess of 20 degrees Celsius may contribute
to the loss of some primary grape aromas and desirable esters and lead to the
production of undesirable higher alcohols (Peynaud, 1981). Some research has
indicated that ester production is strongly influenced by yeast strain. Bisson indicated a
ten-fold difference in ester production could be attributed to strain differences. Most
research indicates that ester production is mainly controlled by temperature (Bisson,
2001). In general, these low molecular weight compounds are retained at cooler
temperatures and lost at higher temperatures.           Therefore, if retention of fruity
characteristics (i.e. esterification of short-chained fatty acids) developed during the
fermentation is desired, fermentation and storage temperatures should be kept fairly
cool.

Research indicates that there may be ideal temperature ranges at which to ferment and
store white wines that results in both increased production and retention of yeast
volatiles. The Umptanum Winery, a 10,000 case facility in the Yakima Valley of
Washington State, was interested in determining an ideal temperature that allowed for
both a clean fermentation and maximum retention of volatiles in their Sauvignon blanc.
Based on the viability and dominance of Saccharomyces at „fairly‟ cool temperatures
and the volatility of aromatic compounds at higher temperatures, the winery postulated
that this ideal temperature might fall in the 15C range. Umptanum‟s production staff also
believed that the post fermentation storage temperatures for the wine could be
substantially lower, just above freezing at 0-5C. The intent of this experiment was then
to assist this winery defining an appropriate temperature range at which to ferment and
subsequently store their Sauvignon blanc. The experiment consisted of fermenting the
wine at three different temperatures (all fairly low) and evaluating both the health of the
fermentation and sensory characteristics of the finished wines.

The owner of the winery heard rumors that cool fermentations have the ability to induce
extremely positive aromatic results in white wines. While I agreed with him, I cautioned
him that fermenting at excessively low temperatures (less than 15 degrees Celsius)
could lead to problems. I made him aware of substantial research relative to my
concerns and suggested that some problems might arise during the experiment. To be
more precise, the temperature in one of the fermentation tanks was be maintained at 12
degrees Celsius increasing the risk of a sluggish fermentation. I even referred him to a
fine book where Peynaud indicated that initiation of a fermentation below 13 or 14
degrees Celsius is nearly impossible (Peynaud, 1984).          We proceeded with the
experiment as planned.

Materials and Methods

To help the winery define an ideal fermentation temperature for its Sauvignon blanc, we
chose to ferment the wine in three separate lots, each at different temperatures (12, 15
and 18 degrees Celsius). These lots shall hereinafter be referred to as lots 12,15 and
18. After the fermentation, all the wines were cold stabilized then stored at freezing to
maximize retention of volatiles. The following outlines the methods by which the winery
conducted and monitored the production of this experimental wine.

Three tons of Sauvignon blanc grapes were hand harvested from Umptanum‟s
vineyards at 23 degrees Brix on September 21, 2001. Their vineyards are located in
the Yakima Valley Appellation, Washington State and were planted in 1991. Upon
arrival at the winery, the grapes were loaded into a stainless steel hopper where sulfur
dioxide was added at 35 ppm. The grapes were subsequently auger fed into a
Healdsburg crusher-destemmer and a Healdsburg reciprocating piston pump was used
to transfer the must directly to a Willmes membrane press (four ton capacity). To
minimize the influence of suspended material on ester formation (Kinzer and Schreier,
1980), the must was gently pressed and the juice then transferred in homogenous form
into a stainless steel tank for gravity settling. This homogeneous press measured 23.5
Brix at 16 degrees Celsius.

After 24 hours of gravity settling, the juice was racked off in equal volumes into three
stainless steel tanks. These stainless fermentation tanks were fitted with floating lids
and temperature controlling jackets. These tanks were chosen to limit the exposure to
oxygen and allow for variable temperature control during the fermentation. The
temperatures for these three tanks were adjusted immediately after the juice had been
pumped in. At the time of inoculation, the tank temperatures were set to three different
values, 12, 15 and 18 degrees Celsius.

On September 22, 2001, the Sauvignon blanc juice was inoculated with Premier Cuvee,
a relatively predictable strain of Saccharomyces byanus. The yeast was added at a rate
of 2% by volume. Juice samples were also collected at this time and analyzed for total
and free amino nitrogen via spectrophotometric assay. Analytical results indicated a
slight deficiency and the juice was appropriately adjusted to 120ppm NH 3 via addition of
diammonium phosphate.

The progression of the fermentation was simply monitored by measuring the reduction
in degrees Brix using hydrometers. The three separate tank temperatures were
maintained throughout the fermentation. The goal was to permit all three wines to
ferment to almost complete dryness at 0.5% RS, and the final residual sugar value was
determined using Clinitest tablets. The temperatures used during the fermentations
were low enough to inhibit most bacteriological activity; however, adjustment of the
molecular SO2 concentration to 0.8 following the ethanol fermentation limited the
possibility for occurrence of the malolactic fermentation. In addition, reduction of the
tank temperatures to 0 degrees Celsius to arrest the fermentation and initiate potassium
bitartrate stabilization also inhibited microbiological activity.

Temperatures were held at freezing for two weeks to ensure cold stability. Samples
were also collected from the tanks at this time and evaluated for protein stability.
Protein stability bench trials indicated the same patterns of haze formation in each wine.
Based on the results of the test, bentonite was subsequently added to each wine at the
rate of 0.5lb per 1000 gallons. Following completion of protein and cold stabili zation,
the wines were clarified using a Seitz Plate and Frame Filter. Sterile membrane (0.45-
micron filter) filtration was performed just prior to bottling.

To simulate realistic conditions, the wines were cellared for six months prior to
sampling. The wines were stored at various temperatures. Each lot (12, 15 and 18)
was divided into sublots and stored at three different temperatures: 10, 15 and 20
degrees Celsius. The wines were opened on May 1, 2001. The method used to
determine the most appropriate temperature with respect to organoleptic quality was
restricted to sensory analysis.
Results

The data for the Brix readings collected from the three separate fermentations is
illustrated in the figure below. As anticipated, the fermentation rates varied according to
temperature. The 12, 15 and 18 degree Celsius lots progressed at rates of 0.58, 0.78
and 0.98 degrees Brix per day, respectively.


                                              Fermentation Curves

                            25

                            20
             Degrees Brix




                            15

                            10

                            5

                            0

                            -5     00


                            10 00

                                   00


                            10 00

                                   00


                            10 00

                                   00
                             9/ 0

                                     0


                             9/ 0

                                     0


                             10 0

                                     0


                             10 0

                                     0

                                     0
                                  /0

                                  /0

                                  /0

                                  /0

                                  /0

                                  /0

                                  /0

                                  /0

                                  /0

                                 0/

                                 2/

                                 4/

                                 6/

                                 8/

                                 0/

                                 2/
                               22

                               24

                               26

                               28

                               30

                                /2

                                /4

                                /6

                                /8

                              /1

                              /1

                              /1

                              /1

                              /1

                              /2

                              /2
                             10




                             10
                             9/




                             9/




                             9/




                            10

                            10




                            10




                            10
                                                           Time

                                             Brix 12 C      Brix 15 C      Brix 18 C



       Figure 1 (above) illustrates the three separate fermentations for the Umptanum Vineyards Sauvi gnon blanc.

The Premier Cuvee fermentation rates corresponded fairly well to historical research
information. Review of the curves above indicates that lot 18 proceeded most rapidly; it
had a fairly short lag time (approximately three days) and the ferme nt was completed in
nearly 24 days. The highest average rate for the Premier Cuvee occurred in lot 18 at a
rate of 0.98 degrees Brix per day. The lot 18 fermentation was healthy and there was
no development of odors indicative of spoilage.

As expected, the fermentation rates for the 15 and 12 degree Celsius substrates were
slower at 0.78 and 0.58 degrees Brix per day, respectively. Lot 15 proceeded normally,
although the lag time was slightly longer than that of lot 18. By the end of the
fermentation, the residual sugar for lot 15 was hovering around 0.5 percent. The figure
above indicates that the lot 12 fermentation had a turbulent beginning and difficulty
reaching dryness. While there was no development of unfavorable odors indicative of
microbial activity, the Premier Cuvee was unable to successfully ferment the reducing
sugars in lot 12, which posed a problem.

After the fermentation, all three lots were cold stabilized and held at 0 degrees Celsius
for two weeks. This procedure also served to ensure retention of volatile compounds
developed by the fermentation. There was some concern with the residual sugar
remaining in lot 12 prior to bottling. However, the sterile filtration conducted at bottling
helped to minimize the risk for microbiological instability. Once lots 12, 15 and 18 had
been bottled, a portion of each was stored at 10, 15 and 20 degrees Celsius.

The wines were opened and evaluated last month, May 2001. A tasting panel
consisting of both experienced and inexperienced tasters was developed. While the
wines were tasted, the focus on the experiment was restricted to development and
retention of fermentation bouquet and varietal aroma. Therefore, the organoleptic
evaluation discussed in this paper only includes these aromatic characteristics. The
following table summarizes the aromatic characteristics determined by the panel of
judges. Each sample was scored on a scale from 1 to 10 (lowest to highest) based on
aromatic intensity.


    Wine Sample                                    General Consensus
  Lot 12 – 10 Celsius     7.0: Lack of balance. Grassiness too overpowering; not enough
                          fruity bouquet. Presence of unpleasant
  Lot 12 – 15 Celsius     7.0: Lack of balance. Grassiness too overpowering; not enough
                          fruity bouquet.
  Lot 12 – 20 Celsius     6.0: Herbaceous. Flat in terms of fruity bouquet.
  Lot 15 – 10 Celsius     10.0: Great balance between classic, grassy aroma and tropical
                          fruity bouquet.
  Lot 15 – 15 Celsius     9.0: Pleasant, grassy varietal character with nice tropical fruit
                          bouquet.
  Lot 15 – 20 Celsius     8.5: Pleasant, grassy varietal character with subtle tropical fruit.
  Lot 18 – 10 Celsius     9.0: Pleasant, grassy varietal character with nice tropical fruit
                          bouquet.
  Lot 18 – 15 Celsius     8.5: Pleasant, grassy varietal character with subtle tropical fruit.
  Lot 18 – 20 Celsius     8.0: Classic varietal aroma with slight hint of tropical fruit.


Discussion

The results for the health of the fermentations and the organoleptic quality of the
Sauvignon blanc produced at different temperatures corresponded well with the
expected outcome and historical research information. As anticipated, utilization of the
three separate fermentation temperatures (12, 15 and 18 degrees Celsius) played a
significant role in the growth rate for Saccharomyces byanus. In addition, these fairly
cool fermentation temperatures encouraged the retention of volatiles produced by
Saccharomyces byanus during the fermentation. However, when the fermentation
temperature was driven below 15 degrees Celsius, Saccharomyces byanus had
difficulty completing the fermentation, which negatively impacted the organoleptic
quality of the wine. These results were similar to those achieved by Killian and Ough,
who determined that lower fermentation temperature (to 15 C) enhances ester
production, but that reducing the temperature to below 15 degrees Celsius does not
necessarily lead to further ester development (membrane (Boulton, Singleton, Bisson
and Kunkee, 1998).

In the Umptanum Winery experiment, the Premier Cuvee was chosen due to its
predictable fermentation characteristics. Consequently, there was little difficulty
isolating the results for the main variable in the experiment, temperature. The results
for the Lot 12 fermentation (12 degrees Celsius) indicated that Saccharomyces byanus
experienced difficulty catabolizing all the reducing sugars at such a low temperature.
The cause for this struggle could have been the result of interference with competing
organisms that have the ability to operate successfully at lower temperatures, such as
Klockera apiculata. Viability of such wild, non-Saccharomyces yeast early in the
fermentation may lower the nutrient substrate available to Saccharomyces and lead to a
reduction in its ethanol tolerance and an incomplete fermentation. To reduce the
potential for such an occurrence, the sulfur dioxide dosage could be increased to a level
that is inhibitory to wild yeast. However, the combination of low temperature and
potentially elevated sulfur dioxide concentration could also reduce the viability of
Saccharomyces byanus. The most appropriate course of action might then be to avoid
attempting fermentations at such low temperatures.

The temperatures in Lots 15 and 18 did not substantially compromise the viability of the
Premier Cuvee in Lots 15 and 18 in comparison to Lot 20. However, a difference in
fermentation rates was clearly experienced at the 15 and 18 degree Celsius levels. The
longer lag period and fermentation time for Lot 15 versus Lot 18 can be explained by
the fact that lower temperatures lower the metabolic activity of the yeast. It should be
noted that the Premier Cuvee did ferment the wine to the desired level of 0.5 percent
residual sugar in both cases, at 15 and 18 degrees Celsius.

The various fermentation and storage temperatures also complicated the sensory
results (restricted to olfactory). The Lot 12 wines all scored lower in quality than the lot
15 and 18 wines, regardless of the storage temperature. The general result with Lot 12
was one where there was little to no development of desirable „fruity‟ fermentation
bouquet, and the varietal character akin to Sauvignon blanc was too pronounced as a
result. Even so, storage temperature did impact the retention of esters in Lot 12 to a
small degree as the sample cellared at 20 degrees Celsius was completely devoid of
fruity bouquet whereas the 10 and 15 degree Celsius samples retained the little bouquet
that had developed during the problematic fermentation.

The wines that were fermented at temperatures high enough to maintain yeast viability
but low enough to encourage production and retention of desirable fermentation
characters scored the highest. The Lot 15 sample that was stored at 10 degrees
Celsius received the greatest overall score of 10 on a scale of 10. The notably pleasing
fruity character that developed during the cool fermentation could potentially be related
to hexyl acetate, which exhibits a distinct fruity odor (membrane (Boulton, Singleton,
Bisson and Kunkee, 1998). Its concentration may be higher at lower temperatures due
to changes in yeast biosynthesis patterns and prevention of hydrolysis reactions
(membrane (Boulton, Singleton, Bisson and Kunkee, 1998). A gradual reduction in the
storage temperatures for Lot 15 was accompanied by a gradual reduction in scores,
possibly due to increased loss of low molecular weight volatiles at higher temperatures.
The Lot 18 samples experienced trends fairly similar to those of Lot 15. However, the
results for Lot 18 indicated a less pronounced production and retention of desirable
aromatic qualities. It is possible that the more rapid fermentation both discouraged
volatile production and retention. There was insufficient time for the level of
esterification experienced in Lot 15, as the duration of the ferment was nearly one week
shorter. There may have also been a slightly higher temperature within the tank for Lot
18 accompanied by more entrapment of volatiles in the column of carbon dioxide
leaving tank.

Conclusion

The results of the experiment at the Umptanum Winery add to historical research
postulating ideal ranges at which to maximize both the production and retention of
desirable esters in white wine. In this test using Sauvignon blanc and Premier Cuvee, a
strain of Saccharomyces byanus, the most ideal fermentation temperature was 15
degrees Celsius. At this temperature, ester development was maximized and retention
of volatiles was the greatest. At 12 degrees Celsius, the temperature was too low and
the viability of the Premier Cuvee was compromised. At 18 degrees Celsius, there was
both production and retention of desirable characteristics, however, not to the same
degree experienced in Lot 15.

In wines where retention of desirable volatile characteristics is of great importance,
storage temperature at the winery must be considered. In this experiment, all wines
were stored at various temperatures and evaluated for retention of desirable aromatic
qualities.    All three lots exhibited the greatest retention of desirable aromatic
characteristics when stored at the lowest temperature, which in this experiment was 10
degrees Celsius. This is an extremely crucial factor for a winery to consider if it plans to
produce „esterified‟ wines. First, the winery must keep these wines cold at the
production facility. Second, it may not be wise to sell „esterified‟ wines outside of the
facility as the winery then loses control of storage conditions and losses in quality may
not be the responsibility of the winery. Such losses in quality could ultimately harm the
winery‟s reputation. The outcome of this experiment led the Umptanum winery to plan
for fermentation of their Sauvignon blancs at 15 degrees Celsius, followed by cold
stabilization and storage at freezing. In addition, the winery only plans to market this
wine at their facility.

Although they were not discussed in detail, there are many other factors to consider
with respect to maximizing the production and retention of desirable aromatic qualities
in wine. In this experiment, the grapes were gently pressed and the juice then settled to
reduce the volume of solids that would be present in the juice. Kinzer and Schreier
(Kinzer and Schreier, 1980) found that polyphenols in suspended material in must can
lead to the inhibition of enzymes responsible for ester production. It may, therefore, be
important to use clarified juices that are free of solids when attempting to produce
„esterified‟ wines. Oxygen is also detrimental to the synthesis of esters. In this
experiment, tanks fitted with floating lids were chosen to limit the exposure of the wine
to oxygen. Aside from temperature control, there are surely many other variables to
consider with respect to maximization and retention of desirable esters in white wines.
Those variables, however, are beyond the scope of this report.

References

Boulton, R., Singleton, V. L., Bisson, L. F., Kunkee, R. E., 1998. “Yeast and
Biochemistry of Ethanol Fermentation”. In: Principles and Practices of Winemaking. PP:
173, 178, 179. Gaithersburg, Maryland: Aspen Publishing.

Bisson, L.F. 2001. Lesson 19 – The Flavor and Aroma Compounds of Wine: Course
Notes from Wine Production. UC Davis University Extension.

Charoenchai, C., Fleet, G., Henschke, P., 1998. Effects of Temperature, pH, and Sugar
Concentration on the Growth Rates and Cell Biomass of Wine Yeasts. Am. J. Enol.
Vitic. 49(3): 283-288.

Fleet, G.H., Heard, G.M., Gao, C., 1989. The Effect of Temperature on the Growth and
Ethanol Tolerance of Yeasts During Wine Fermentation. Seventh International
Symposium on Yeasts: S43-S46.

Kinzer, G., Schreier, P., 1980. Influence of Different Pressing Systems on the
Composition of Volatile Constituents in Unfermented Grape Musts and Wines. Am. J.
Enol.. Vitic. 31(1): 7-13.

Peynaud, E., 1984. “Conditions for Development of Yeasts-Conducting Alcoholic
Fermentation. In: Knowing and Making Wine. PP: 107-108. John Wiley and Sons, Inc.

								
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