Assessments of Difficult to Ferment Juices

The goal of this project is to uncover the causes of chronically difficult to ferment juices. These juices often derive from the same vineyard or block of a vineyard and other similarly managed vineyards and blocks display normal fermentation kinetics. These difficult to ferment juices do not appear to respond to nitrogen or other commercial nutrient addition, occur regardless of strain used, and are challenging to restart. Our aims for the first year of this project were to evaluate the nutritional content of these juices to determine if a nutritional deficiency or presence of toxic compound was the cause of inhibition of fermentation. In this first year we were able to narrow down the possible issues with these juices. Although low nitrogen is a factor, addition of nitrogen to the fermentation seems to partially address the nutritional limitation. In addition these juices appear to impose a high vitamin demand on the yeast which we will explore in detail in this second year. This past year we were able to demonstrate that the accumulation of mannitol observed from the metabolomics analysis of the yeast strains grown in difficult to ferment juices is an indicator of oxidative stress in the juice.

It is not clear what juice compositional factors are leading to the oxidative stress and why this is not alleviated by use of Sulfur dioxide, and this will be explored further in this coming year. The low arginine and high proline of these juices suggests a problem with development, activity or destruction of the fine roots of the vine. Addressing this problem by adjustment of either yeast strain or nutrient supplementation would prevent the need for more invasive vineyard management strategies.

In this past year several commercial wineries sent us examples of chronic to ferment juices and we discovered that these juices either resembled the J. Lohr juice in having a deficient nutrient profile or were characterized by the presence of four rare (for grape) acetic acid bacteria species that seem to retain viability throughout the winemaking process and that appear to be inhibitory in ways not due to simple acetic acid production. An added goal for this coming year’s grant is to evaluate the role(s) of these bacteria in fermentation progression and to assess their sulfite sensitivities and persistence during fermentation.

Understanding Alcohol Tolerance in Wine Yeasts

Understanding alcohol tolerance in wine yeasts is important in order to develop tools to rectify sluggish and stuck wine fermentations and new commercial yeast strains with desirable sensory or process characteristics. In previous work, we have identified two potential mechanisms for alcohol tolerance including ones related to the composition of the yeast cell membrane and to the nutrient utilization efficiency of the strain. To understand these mechanisms, we further examined both potential effects. Specifically, we were able to find that the composition of the cell membrane changes significantly as fermentation temperature is raised or lowered. Using our high resolution methods for measuring membrane composition, we were also able to identify specific types of lipids in the cell membrane that are associated with lower ethanol tolerance (e.g. several phosphatidylinositol lipids) and higher ethanol tolerance (e.g. several phosphatidylcholine lipids). In this period, we also developed and used a method for measuring a wide range of metabolites inside and outside of yeast cells during fermentation. Our analysis shows that certain metabolic pathways like the pentose phosphate pathway and glycerol secretion are negatively correlated with cell growth and ethanol tolerance of these strains. These major results have allowed us to begin to identify targets for genetic manipulation of wine yeasts that should increase or decrease alcohol tolerance. We hope to actively initiate this part of the project prior to the completion of funding this year. The work completed as part of this grant has resulted in two papers (one published and the other in final revisions after review) with two more in preparation and a number of presentations at local and national meetings.

Formation of Volatile Sulfur Compounds in Pinot Noir Post-Fermentation -Part 2: Lees Level and Contact Time on Volatile Sulfur Compounds in Wine

During the first year of this project, wine fermentation and aging at various amounts of lees were conducted by Dr. James.P.Osborne as a part of a separate project “Formation of volatile sulfur compounds in Pinot noir post-fermentation. Part 1: Role of grape amino acid content and wine lees composition”. Two yeast strains, and three lee levels for used in the study. Initial samples were collected for amino acids and volatile sulfur analysis. Additional samples were taken after 2 and 4 weeks and 2 months of storage and assessed for the same parameters. The project is still at very early stage, and limited results showed:

  1. The concentrations of volatile sulfur compounds in the wines fermented with both two yeasts were low after fermentation and pressing. All of the major volatile sulfur compounds were analyzed including H2S, mercaptans, disulfides, thiol esters. Saccharomyces cerevisiae P1Y2 produced lower levels of methyl thioacetate and methionol than Saccharomyces cerevisiae RC212.
  2. Heavy lee loading resulted higher level of H2S initially, but the difference diminished after aging. No significant differences were observed for other volatile sulfur compounds at different levels of lees.
  3. After 2 months of aging, some sulfur compounds such as methionol decreased while some sulfur compounds like methyl thioacetate kept consistent.
  4. Some industrial samples were analyzed, and high levels of H2S were detected in some of the wine samples, and industrial partners responded with cupper treatment.

Impact of Malolactic Fermentation on Red Wine Color

The color of a red wine is an important sensory attribute that originates primarily from anthocyanins. However, development of stable red wine color is impacted by compounds such as p-coumaric acid, caffeic acid, catechin, and quercetin that are involved in copigmentation reactions as well as acetaldehyde and pyruvic acid. While it is known that yeast can alter the concentrations of some of these compounds, little is known regarding the impact malolactic bacteria may have on red wine color development. This project investigates the effect of the malolactic fermentation (MLF) on red wine color and the ability of malolactic bacteria to degrade compounds important to the development of stable red wine color. Pinot noir and Merlot wines were produced in 2009. A portion of the Pinot noir and Merlot wines underwent a simultaneous alcoholic and malolactic fermentation (S. cerevisiae VQ15 + O. oeni VFO) while the remainder were only inoculated with S. cerevisiae VQ15. At dryness, all wines were pressed and sterile filtered (0.45 ?m). For both the Pinot noir and Merlot wines, three carboys were inoculated with either O. oeni strain VFO, Alpha (Lallemand), or VP-41 (Lallemand) at approximately1 x 106 cfu/mL. The remaining carboys of wine were not inoculated with O. oeni. All wines were kept at 20°C. Some of the wine that had not undergone MLF was pH adjusted to the same final pH of wines that had completed MLF. Samples were taken before and after MLF for analysis and wines were sterile filtered, bottled, and stored at 13°C. As had been seen with wines produced in 2008, wines that underwent MLF, including wine produced with a simultaneous alcoholic and MLF had the lowest color throughout storage. MLF (+) Pinot noir wines had significantly lower color after completion of MLF (day 0) and this difference remained after 180 days with close to a 20%difference in color @ 520 nm being observed. This trend was also seen for the Merlot wines although the differences were larger with a 30-35%difference in color @ 520nm after 180 days storage. Color differences appear to be related to pigmented polymer formation as after MLF both Pinot noir and Merlot wines had significantly lower pigmented polymers than wines that had not undergone MLF. Differences increased over time and by day 180 there was a 30-35%difference between MLF (+) and MLF (-) Pinot noir wines and a 40%difference for Merlot MLF (+) wines. Furthermore, there were significant differences between the monomeric anthocyanin content of MLF (+) and MLF (-) wines. Pinot noir and Merlot MLF (+) wines contained significantly higher concentrations of monomeric anthocyanins than MLF (-) wines. These differences increased after 90 days storage with Pinot MLF (+) wines having 30-40%greater monomeric anthocyanin content than control pH wines and a 30-48%difference for Merlot MLF (+) wines. Overall, different O. oeni strains did not cause a significant difference in color @ 520 or polymeric pigment indicating that O. oeni strains did not impact red wine color differently.

Non-Saccharomyces Yeast on Wine Quality – Part 2, Aroma and Flavor Development (year 1)

In Fall 2009, fermentations using high hydrostatic pressure (HHP) treated grapes were conducted using grapes from Woodhall vineyard (Alpine, OR). Grapes were harvested at 24.8 °Brix (TA 0.76 g/L and pH 3.58). After HHP treatment, grapes were transferred to sterile micro-fermentors and inoculated with different yeast. There were total 4 different blends of yeasts used in the fermentations, which were S. cerevisiae MERIT.ferm (MS), MERIT.ferm + Kluyveromyces thermotolerans (60:40 ratio) (MK), MERIT.ferm + Kluyveromyces thermotolerans + Torulaspora delbrueckii (60:20:20 ratio) (MKT), and a S. cerevisiae isolated from an active fermentation in a winery that does not inoculate with commercial starter cultures (AR). All fermentors were then placed in a temperature controlled room set at 27_. All of the fermentations completed alcoholic fermentation (sugar < 0.5 mg/L) in less than 144 hrs. After pressing, wines were allowed to settle for 48 hours at 4_, sterile filtered through a 0.45 um cartridge filter, bottled and stored at 13__ until analysis (GC-MS analysis occurred 10 months post-pressing). Each treatment has 3 replica, so total 12 wine samples were studied.. The result demonstrated that the volatile compounds were very similar in the four yeast treatments but the concentrations varied a lot. MKT treatment had the highest concentration of ethyl esters of acetate, butanoate, hexanoate, octanoate, and decanoate, as well as ethyl vanillate. However, both MK and MS treatments had higher concentration of branch-chained esters such as isoamyl acetate, ethyl 2-methylbutanoate and ethyl 3-methylbutanoate . In addition, fermentation yeasts showed an effect on terpene alcohol concentration. MKT treatment had the highest level of geraniol. The yeast treatment did not show impact on the concentration of linalool, citronellol and ?-damascenone. The newly isolated non-Saccharomyces yeasts from local wineries will be evaluated in 2011.

Understanding and Increasing Alcohol Tolerance in Wine Yeasts

The goal of our project is to measure the cell membrane composition of a wide range of wine yeasts and correlate this composition with strain characteristics such as growth and ethanol tolerance, thus developing a rational approach to strain modification. In this way, new strains can be constructed that are highly ethanol tolerant or ethanol tolerance can be introduced into existing strains with favorable flavor characteristics. To achieve these goals, we first identified a group of yeast strains representing a wide range of ethanol tolerances. We then did an in-depth examination of the growth characteristics and ethanol tolerance of these strains, and confirmed that we do, in fact, have a wide range of characteristics. We have completed development of a new HPLC-mass spectrometer (LC-MS) assay for determining cell membrane lipid composition. The main classes of lipids in yeast membranes are well separated with this method, and we are able to quantify over 80 species of fatty acids making this significantly higher resolution than the traditional methods for lipid analysis that have been used previously in studies on Saccharomyces. We are nearly finished with the associated assay for sterols. We used this assay, in its current state, to examine the membrane composition of six of the strains with fairly different ethanol tolerances from a point during small scale fermentations. These data were used, along with multivariate statistical approaches (e.g. Principal Component Analysis), to begin to examine how membrane composition varies with fermentation properties and ethanol tolerance levels. Studies to examine yield of cells from nitrogen were also initiated.

Adaptive Evolution of Commercial Wine Strains for Reduced Ethanol

The goal of this research project was to explore the use of the impact of chronic exposure to a moderately inhibitory concentration of furfural under fermentative conditions on cellular metabolism. Furfural inhibits yeast metabolism and therefore growth by competing with acetaldehyde for the reduced cofactor, NADH, generated during sugar catabolism. The reaction of acetaldehyde with NADH produces ethanol. Similarly the reduction of furfural produces a much less toxic alcohol. The yeast cell is therefore faced with a challenging metabolic problem: in order to detoxify furfural in the environment the cells must make less acetaldehyde and therefore less ethanol. Adaptive evolution is a process that exposes yeast to chronic inhibitory concentrations for multiple generations thereby imposing selective pressure to mutate to become more resistant to the inhibitory conditions. The first aim of this project was to screen a collection of commercial wine strains for tolerance to furfural and 5-hydroxymethylfurfural. We expected higher tolerance given that furfural is found during barrel fermentation and aging and resistance to this compound would be expected across wine strains. All 27 of the wine strains tested displayed some level of resistance to furfural in contrast to the 3 laboratory strains that displayed sensitivity. Upon more detailed analysis of sensitivity, the wine strains were grouped into six clusters depending upon their patterns of resistance. Ethanol yields were evaluated for strains in each of the clusters and one cluster appeared to contain strains that were low ethanol yielders, validating this approach as a mechanism to generate strains with reduced ethanol yields. Adaptive evolution experiments are underway to hopefully lead to even further reductions in ethanol production.

Understanding and Increasing Alcohol Tolerance in Wine Yeasts

The goal of our project is to measure the cell membrane composition of a wide range of wine yeasts and correlate this composition with strain characteristics such as growth and ethanol tolerance, thus developing a rational approach to strain modification. In this way, new strains can be constructed that are highly ethanol tolerant or ethanol tolerance can be introduced into existing strains with favorable flavor characteristics. To achieve these goals, we first identified a group of yeast strains representing a wide range of ethanol tolerances. We then took a 12-member subset of this group and did a preliminary examination of the growth characteristics and ethanol tolerance of these strains, confirming that we do, in fact, have a wide range of characteristics. We have nearly completed development of a new HPLC/mass spectrometer (LC/MS) assay for determining cell membrane composition, and are in the process of moving this assay over to a more sensitive instrument for quantification. The main classes of lipids in yeast membranes are well separated with this method. We used this assay, in its current state, to examine the membrane composition of two strains with fairly different ethanol tolerances at several points throughout small scale fermentations. We demonstrated that membrane composition does, in fact, change over the course of fermentation, and is also quite different for the different strains. In addition, our initial experiments have shown that ethanol tolerance may be a dual function of both membrane composition and nitrogen utilization efficiency.

Impact of malolactic fermentation on red wine color

The color of a red wine is an important sensory attribute that originates primarily from anthocyanins. However, development of stable red wine color is impacted by compounds such as p-coumaric acid, caffeic acid, catechin, and quercetin that are involved in copigmentation reactions as well as acetaldehyde and pyruvic acid. While it is known that yeast can alter the concentrations of some of these compounds, little is known regarding the impact malolactic bacteria may have on red wine color development. This project is investigating the effect of the malolactic fermentation (MLF) on red wine color and the ability of malolactic bacteria to degrade compounds important to the development of stable red wine color.

Pinot noir and Merlot wines were produced using grapes from the Oregon State University vineyard and were fermented with Saccharomyces cerevisiae VQ15. Concurrently, a third of Pinot noir musts were inoculated with Oenococcus oeni. At dryness, wines were pressed and filtered (0.45 µm nominal) with a second third of the wines being inoculated with O. oeni VFO (remaining third was was not inoculated). Some of the wine that had not undergone MLF was pH adjusted to the same final pH of wines that had completed MLF. Samples were taken before and after MLF for analysis and wines were sterile filtered, bottled, and stored at 55° F.

Prior to MLF, all Pinot noir wines had very similar concentrations of acetaldehyde and pyruvic acid. This was also the case with the Merlot wines. However, all wines that had undergone MLF, including the simultaneously fermented Pinot noir wine, had lower acetaldehyde and pyruvic acid concentrations. Pinot noir wines that had undergone MLF also had lower wine color and polymeric pigment values compared to wines that had not gone through MLF. For the Merlot wines, wines that had undergone MLF also had lower wine color, copigmentation, anthocyanins, and polymeric pigment than wines that had not undergone MLF with the differences being more pronounced then what was observed for the Pinot noir wines. The phenolic composition of wines that underwent MLF was different from wines that had not. Both Pinot noir and Merlot wines that had undergone MLF had lower levels of caftaric acid and higher levels of caffeic acid than wines that had not undergone MLF. Wines that did not undergo MLF also had lower malvidin glucoside and monomeric anthocyanin concentrations than wines that had undergone MLF. Finally, the concentration of tannin in Pinot noir wines that had undergone MLF was lower than in wines that had not. These results demonstrate that MLF as well as time of bacterial inoculation can effect the concentration of phenolic and non-phenolic compounds involved in red wine color development.

Interactions Between Nitrogen and Vitamins on Fermentation Rate and H2S Production by Saccharomyces

Sluggish fermentation and hydrogen sulfide production are currently serious problems facing the wine industry. Besides nitrogen deficiency, a lack of certain vitamins such as thiamine and pyridoxine, can also impact H2S formation, coenzymes involved in yeast metabolism, play an important role in sulfur production. A comprehensive and systematic research approach was conducted to determine how nitrogen and vitamins (thiamine and pyridoxine) influences yeast growth rate, fermentation rate, and hydrogen sulfide production. Synthetic grape juice based on the amino acid composition of Cabernet Sauvignon grape must was used for fermentation was inoculated with Saccharomyces cerevisiae strain Montrachet (UCD 522). Fermentation rate (decrease in soluble solids), yeast viability, and H2S production were all affected by the availability of nitrogen and these vitamins.