Protracted or stuck fermentations are a recurring problem in the wine industry. Nutritional supplementation often stimulates fermentation and can reduce the incidence of problem fermentations. Fermentation rate decreases as a consequence of a drop in sugar transport capacity. Sugar transporters are the key site of control of flux through glycolysis. The goal of this research program is to determine which glucose transporters are expressed during grape juice fermentation and to define the mechanisms of regulation of transporter activity. In addition, we are also investigating nutritional and environmental factors that affect fermentation rate. Last year we discovered that a suboptimum potassium to proton ratio results in a sluggish fermentation. Potassium is know to be stimulatory to fermentation rate and it has been postulated that this is a cell surface phenomenon, that is, that potassium directly stimulates glucose transport. Given that some rootstocks now used commercially in California result in low petiole potassium, we proposed investigating the effect of the potassium to proton ratio in natural juices on fermentation rate using commercial yeast strains. We also explored the effect of other ions on fermentation rate. We discovered that the potassium effect is not mediated at the level of the cell surface, and that once the fermentation is stuck, addition of potassium is unable to re-initiate fermentation. Re-inoculation also does not re-initiate fermentation even with the addition of potassium.
The formation of hydrogen sulfide during fermentation was followed by direct headspace sampling and quantification by gas chromatography with flame photometric detection. The formation of acetic acid was followed in the same fermentations but by sampling and capillary electrophoresis. Juice samples were analyzed for individual amino acids and correlations between these values, calculated free amino nitrogen (FAN) content and the formation of acetic acid and hydrogen sulfide were performed. The formation of acetic acid was very low (mean-0.09 mg/L, sd = 0.10 mg/L.) and could not be correlated with any of the compositional factors. The formation of hydrogen sulfide was variable (mean = 37 ug/L of juice, sd = 64 ug/L), ranging from none to 300 ug/L. The effect of yeast strain showed a wide variation in a single juice (mean = 226 ug/L, sd = 128 ug/L) with the cultures of Fermevin, Prisse du Mousse, Premier Cuvee being below the mean and those of Montrachet, Pasteur Champagne, Enoferm ICV-D46 and Lavin 7IB being above the mean. In the temperature series, the production of hydrogen sulfide increased from 68 ug/L at 15°C to 164 ug/L at 30°C and then fell to 56 ug/L at 35°C. Statistical analysis (principal component analysis) found the formation of sulfide during fermentation to be positively correlated (CC=+0.56) with the FAN content and (CC=+0.61) with the total nitrogen content. With respect to individual amino acids, alanine (CC=+0.72), glutamine (CC=+0.71), gamma-amino butyric acid (CC+0.68), glycine (CC=+0.66) and methionine (CC=+0.63) were the most strongly correlated with sulfide formation. These results with California juices are contrary to the widely-held belief that sulfide production is caused by low levels of free amino nitrogen.
In this grant year, the investigation of the genetic and environmental factors that influence fermentation rate was continued. A more detailed analysis of the effect of limitation of individual amino acids was conducted. This analysis revealed that yeast cells are particularly sensitive to lysine limitation, moderately sensitive to adenine and leucine levels, but fairly insensitive to tryptophan, histidine and uracil concentrations. The levels of an amino acid that support maximal growth rate seem to be lower than the concentrations required for maximal fermentation rate, for some but not all nitrogen sources. In addition to analysis of nitrogen levels and reduced fermentation rate, we also discovered that an imbalance of potassium to hydrogen ion concentration would lead to a stuck fermentation. Maximum fermentation rate is equivalent in low pH juices and in juice-like synthetic media of differing potassium ion concentrations. However, in low potassium juices stuck fermentations develop. Sugar transporters, the HXT proteins, are degraded under conditions leading to arrest of fermentation. Analysis of degradation of one of these transporters, the HXT2 protein, revealed dramatic instability. This protein is degraded rapidly upon cessation of growth and upon nutrient limitation. There are now 14 HXT genes known in Saccharomyces. Not all of these transporters is expressed during grape juice fermentation. We are still in the process of determining which ones are expressed under these conditions.
Commercial yeast and lactic acid bacteria (LAB) starter cultures are commonly used to promote the alcoholic and malolactic fermentations (MLF) in winemaking. MLF is especially important in cool climate grape growing regions (e.g. New Zealand) where high acid wines are produced. MLF reduces acidity, as malic acid is decarboxylated to the weaker lactic acid. Wine yeast and LAB are not necessarily neutral in association and can interact in vinifications. For example, the yeast can cause inhibition or stimulation of the LAB which may result in delay or promotion of MLF. The aim of this research was to screen commercial yeast strains for their inhibitory or stimulatory action on wine LAB using an agar diffusion assay. Subsequently, observed interactions were confirmed in actual grape juice fermentations, and the timing and mechanism of inhibition/stimulation during a fermentation was investigated.
Many environmental and physiological factors influence fermentation performance of Saccharomyces. Nitrogen or oxygen limitation result in sluggish or stuck fermentations, albeit for different reasons. Other factors are also important and interactive. For example, a micronutrient (minerals or vitamins) limitation may exacerbate a nitrogen shortage. Often these interactive effects are not predictable from studies of the effect of the factor in isolation. Our goal is to go beyond empirical observations and studies of one variable in defined media to understanding the biochemical basis of the yeast response. Sugar transport is the site of action of factors affecting fermentation performance such as nitrogen or nutrient limitation, yeast strain, phase of growth, temperature and possibly pH. Transport of sugars in Saccharomyces is complex and a function of the activity of different transporter proteins working in concert. The timing and level of expression of these proteins can be manipulated. In this research program, we are manipulating the level of activity of individual transporters via mutation and over expression, and assessing the effect of that genetic change on ability to complete a grape juice fermentation. Our focus is on the regulation of sugar transport by nitrogen limitation so that eventually yeast strains can be engineered that will complete a fermentation regardless of the nitrogen signal.
The quantity as well as quality of available nitrogen during fermentation of grape juice appears to affect the rate of fermentation. If too high, rapid fermentation occurs generating excessive heat that may pose a problem or require temperature control. If too low, nitrogen limitation will result in a sluggish or even stuck fermentation. The site of control of fermentation rate is uptake of the sugar into the cell. Sugar uptake is mediated by specific transport proteins located in the plasma membrane at the cell surface. Five glucose transporter genes have been identified in my laboratory. The role of each of these genes in anaerobic grape juice fermentation is being analyzed. Simultaneous loss of two of these genes (SNF3 and HXT1) resulted in a yeast strain completing grape juice fermentation under conditions where the wild type parental strain yielded a stuck fermentation. This finding has important implications for the genetic engineering of a yeast strain for wine production that will be less likely to stick or fail to complete fermentation. In addition, it was found that adenine concentration can be stimulatory to fermentation increasing the maximum fermentation rate and decreasing overall time to dryness. The adenine effect was not a simple consequence of extra nitrogen, as supplementation with the same or higher levels of non-adenine nitrogen compounds did not result in the same stimulation. However, the adenine effect was both strain and temperature of fermentation specific. There was a greater effect with Montrachet than with Prise de Mousse or Pasteur Champagne, and a greater effect at warmer temperatures (20 versus 15°C). The effect of timing of addition of adenine on fermentation performance was complex, requiring additional studies to evaluate properly.
SUMMARY AND RESEARCH ACCOMPLISHMENTS: Preliminary results indicate that although there is indeed an increase in the aroma/bouquet of the wines whose volatiles have been added back, some of the panelists agreed in that “the increased aroma and bouquet do not necessarily translate into an increase in quality of the wines.” This was particularly true for white wines fermented at 80 deg F compared to those fermented at ’55 deg F.
Adenine supplementation stimulated fermentation rate in Montrachet at low (15°C) temperature by reducing the lag time to onset of fermentation, and by shortening the time to dryness. There appeared to be little impact on maximum fermentation rate. Prise de Mousse and Pasteur champagne were unresponsive to adenine supplementation. The effect of adenine was greatest at an intermediate concentration and not affected by nitrogen supplementation. Thus, the stimulation appears to be adenine-specific, not a simple consequence of the presence of extra nitrogen. Loss of two glucose transporter proteins SNF3 and HXT2, reduced rate of fermentation at the end of fermentation. The HXT1 glucose transporter does not play a major role during vinifcation. Interestingly, fermentations conducted by mutants lacking HXT2 were always over run with bacteria. Loss of this gene affected competitiveness of this yeast strain.