Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae
In this current grant year, it was discovered that some of the variability in response to phenolic compounds displayed by different commercial strains was possibly due to subtle differences in preparation of the synthetic juice medium, Triple M. This was confirmed using slightly different protocols and the Premier Cuvee yeast strain. Therefore, the preparation and composition of this Medium has been redesigned this year in order to avoid this problem. Analysis of the impact of phenolic compounds is continuing. To date, gallic acid appears to be uniformly stimulatory at concentrations found in wine while ferulic acid may be neutral, stimulatory or inhibitory to sugar consumption under laboratory conditions mimicking natural fermentations. The hexose transporters encoded by the FDTT9/HXT11 genes that are under multidrug resistance control were shown to be expressed under enological conditions. The presence of these genes has been shown to be stimulatory for the uptake of inhibitory drugs. They may therefore play a role in uptake or release of phenolic compounds inside of the cell by coupling such movements to the uptake of glucose. In this current grant year, we launched the comparative analysis of the impact of phenolic compounds on different yeast strains (conducted by Laura Lange). She initially found that the effects were quite variable and there appeared to be no consensus as to whether the effects of a given compound were stimulatory or inhibitory with the exception of gallic acid. In the course of her work, we discovered that the inherent variability in the composition of the Triple M medium led to variation in the response of the yeast. The pH of this medium is adjusted using ammonium hydroxide. We found that since the pH of our water supply varied, so did the ammonium content of the medium. Further analysis revealed that this difference in nitrogen content was not impacting the results. However, the medium has been redesigned to contain a constant concentration of nitrogen. Another problem with the medium was the low concentration of potassium. Previous work in my laboratory indicated that potassium deficiency could lead to a sluggish fermentation and that this effect was somewhat strain dependent. We therefore decided to use potassium hydroxide to adjust the pH of the medium to assure a reasonably high concentration of potassium. Finally, the micronutrients were prepared as a stock solution and diluted when added to the medium. We noticed that a precipitate formed in the stock upon storage and that the age of the stock solution was correlated with variability in the effect of the phenolic compounds. This makes sound physiological sense since the cofactors derived from the vitamins and minerals in this mix are critical for respiratory activity of the yeast. To eliminate this problem, we now make the micronutrient cocktail fresh each time and use the Triple M medium within 48 hours of preparation. This has eliminated the variability in the response. This important optimization of the medium slightly delayed progress on the grant, but we are now conducting the comparative studies across strains. This will not be finished as originally hoped by the end of this grant year, so will continue into the first three months of the next grant year.