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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