Managing Protection of Varietal Aromas From Wine Oxidation

In order to investigate the reactions of quinones with unknown nucleophiles to further understand how quinones react in wine. The quinone reaction products are investigated by Q-TOF using 13C6 labels. Since the labeled compound is expensive, we used the unlabeled catechol (12C6) first to determine the levels of 13C6 labels we needed, the incubation time, and the Q-TOF method development. We have set up a list of the products from quinone with the known nucleophiles and optimized the analysis method to maximize the numbers of the detection of these known products. Considering the total amount of catechols, such as caffeic acid, catechin, cyanidin, are around 2g/L for red wines and 0.5g/L for white wines and the expecting detection limit of the product is around 2 mg/L, 0.1g/L quinone was added to wines and the level was confirmed by the trials. The incubation time was also tested and finally chosen as 2 hours.

Assessments of Difficult to Ferment Juices

The goal of this project is to uncover the causes of chronically difficult to ferment juices. These juices are often derived from the same vineyard or block of a vineyard and othersimilarly managed vineyards and blocks displaying normal fermentation kinetics. We haveconfirmed the inhibitory role of previously identified lactic acid bacteria in yeast fermentation buthave also discovered that these bacteria are efficient at inducing the establishment of the GAR+prion in wine strains. This prion is a protein conformational change that is inherited by progenycells during cell division, thus once cells in the population have changed to establish the prion subsequent generations will also be in the prion state without need for continued induction. We have also discovered that commercial wine strains that rapidly induce the prion as this induction occurred at a rapid rate in five of the 11 genetically unrelated commercial wine strains evaluated. The prevalence of this ability to rapidly induce this state suggests the prion state plays an important role in survival during wine fermentation. We are continuing to work out the metabolic changes that occur under these conditions to help identify or genetically construct via breeding strains that would be insensitive to the bacteria but also able to grow and ferment normally. In addition to inhibitory lactic acid bacteria, we discovered three species of acetic acid bacteria that can be found on grape berries at harvest that lead to arrest of fermentation. One of these bacteria, Gluconobacter cerinus, is as efficient as the lactic acid bacteria in inducing formation of the GAR+ prion. The other two acetic acid bacteria, Acetobacter malorum and Acetobacter ghanensis are inhibitory to yeast growth, showing similar levels of inhibition as Acetobacter aceti but without making the high concentrations of acetic acid found with A. aceti infection of wine. The inhibitor in this case is as yet unknown. Under certain metabolite conditions of low nitrogen or low vitamins we have shown that the high proline can be inhibitory to yeast metabolism.

The metabolomics analyses performed this past year confirmed the presence of mannitol in sluggish fermentations from this fruit and confirmed the induction of mannitol accumulation in juices by treatment with oxidizing agents. Our working model is that the phenolic profile of these juices may have changed due to environmental conditions and the accumulation of proline in the berry is done to minimize an inhibitory aspect of this compound or compounds and that yeast cells are similarly sensitive to these inhibitors and accumulate mannitol to minimize inhibition. We further propose that high proline might interfere with the functionality of the mannitol or lead to changes in the cell membrane that decrease ability to take up key nutrients like vitamins thus requiring higher vitamin supplementation.

Tannin Structure-Activity Relation to Red Wine Astringency

Optimizing wine quality with regard to mouth feel is a quest for wineries that are trying to fine tune the astringency of their wines to a desirable level. Consistent with that, various wineries had agreed and participated in the extended maceration project in order to understand the driving force behind astringency. With that in mind, this project was born to comprise different analytical techniques, which are necessary to construct a conclusive understanding of tannin’s activity. During the 2014 vintage, maceration trials were conducted in five Napa Valley wineries. Winery selection was based upon winemaker interest. For each winery, fermentations were set up according the the specific cooperators protocol. At various times during the course of fermentation/maceration, samples were collected following pumpover operations. Following collection, samples were transported and stored at 7 degrees Centigrade until processed. For tannin isolation, each sample was filtered using a 9.0cm Whatman filter paper (20-25 μm pore size), diluted with milli-Q water (50:50 v/v) and then loaded onto a preconditioned column containing Toyopearl chromatography resin (HW 40C). Purification of tannins was conducted according to Aron et al.1 Briefly, and following application to the column, the column was washed with water followed by 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} v/v aqueous methanol and then tannins were eluted with 66{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} v/v acetone in water. The acetone was removed by rotary evaporation and the aqueous portion containing the tannin was lyophilized to a powder. Gravimetric yields were determined and the tannin powders were stored in glass, air tight, vials after being sparged with N2 and stored under -20°C. Tannin isolates underwent analysis using a variety of analytical techniques including gel permeation chromatography, phloroglucinolysis and stickiness. The analysis of tannins’ stickiness has recently been introduced by Barak et al.2 and further developed by Revelette et al.3. Samples were prepared according to the above methods and the enthalpy of interaction was determined for tannin polymers absorbing at 280 and 520 nm. Secondly, tannin powders underwent acid catalyzed degradation in the presence of excess phloroglucinol (phloroglucinolysis) to determine the subunit composition, average degree of polymerization and conversion yield following the method of Kennedy and Jones.

Following phloroglucinolysis, tannins were analyzed by gel permeation chromatography which provided information on size distribution5. Across all experiments, a total of 104 tannin isolates were prepared. Given that these isolates represent tannins collected at various points during fermentation and maceration and across different blocks and varieties, it is believed that a realistic picture of commercial structure variation for Napa Valley has been achieved. Results to Date are as follows: Winery 1 investigated the effect of extended maceration on tannin composition and activity. One Cabernet sauvignon block was harvested and equal portions of fruit were contained in two identical fermentation vessels. The must underwent a prefermentation soak for four days before inoculation. Samples for the control tank were collected on a semi-daily basis until press day (total soak time was 15 days). On the last day (day 15), the first extended maceration sample was collected. Similarly, extended maceration samples were taken on a semi-daily basis until press day (total soak time was 26 days).

Formation of Volatile Sulfur Compounds in Pinot Noir Post-Fermentation

Development of volatile sulfur compounds (VSCs) post-fermentation can be a significant issue during both red and white winemaking. Unfortunately our understanding of contributing factors or conditions that impact VSCs is limited due in part to the complexity of their formation. This study focuses on the development of VSCs in Pinot noir during post-fermentation aging. During the first year of the study the impact of lees levels and composition on formation of VSCs was determined. Results showed that although lees levels and yeast strain impacted the amount of sulfur containing amino acids (pre-cursors for the formation of volatile sulfur compounds) in the wine, this did not result in an increase in the formation of volatile sulfur compounds. Wine samples were also provided by collaborating wineries in 2013 and assessed for VSCs so as to determine the cause of early reduction issues in barrel. Wineries were instructed to take juice and wine samples from lots that traditionally had issues with VSCs. Wine samples were taken after pressing and after one, three, and nine months barrel aging. Analysis of these samples indicated that the early formation of reductive smells soon after going to barrel were most likely due to H2S rather than the formation of more complex volatile sulfur compounds such as mercaptens and disulfides. Where this H2S is derived from and what factors impact its formation became the focus of future experiments. Firstly, experiments investigating the role of YAN concentration and content were undertaken. A synthetic grape juice was prepared where the amount and type of YAN (primary amino acids vs. ammonia from diammonium phosphate (DAP)) could be varied. H2S production was measured throughout fermentation and finished wines were assessed for a range of other VSCs. Variation in YAN concentration as well as whether YAN was derived from amino acids or DAP impacted H2S production during fermentation as well as formation of volatile sulfur compounds post-fermentation. In particular, DAP supplementation increased the amount of H2S formed late in fermentation and resulted in the highest amount of methyl thioacetate in the wines post-fermentation. Experiments investigating the role of elemental sulfur in the formation of H2S and other volatile sulfur compounds post-fermentation were also undertaken.

Pinot noir grape fermentations  were undertaken where an addition of 0, 5 or 15 ug/g elemental sulfur was made to the grapes. Fermentations were conducted by a high H2S producing yeast strain (UCD522 ) or a no-H2S producing yeast strain (P1Y2). Addition of elemental sulfur to the grapes resulted in H2S formation during the alcoholic fermentation independent of which yeast strain was used. H2S production was higher in fermentations performed by UCD522 with increasing amounts of elemental sulfur resulting in increased production of H2S. In addition, higher elemental sulfur additions also resulted in higher H2S production late in fermentation. This is particularlyimportant as H2S formation late in fermentation is more likely to be retained in the wine due to the reduced production of CO2 by yeast. Higher elemental sulfur also resulted in wines containing higher concentrations of methyl thioacetate post-fermentation. Both of these findings suggest an important role for elemental sulfur in the formation of volatile sulfur compounds during and after fermentation. Overall, this study to date has demonstrated that lees levels impact the concentration of sulfur containing amino acids in the wine but may not directly impact formation of volatile sulfur compounds. Instead, the formation of H2S late in fermentation or early post-fermentation may be the main cause of post-fermentation reduction soon after wine goes to barrel. Current experiments are investigating the impact of YAN, yeast strain, and elemental sulfur on the formation of H2S and other volatile sulfur compounds post-fermentation. This work includes an ongoing effort to measure the amount of elemental sulfur present on grapes at harvest.

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

The effect of lees contact time during wine barrel aging on volatile sulfur compounds was investigated in this study. Pinot noir wines were made using grapes from the Oregon State University vineyard and fermented with two different commercial yeast strains. One set of wines was produced using the low/no H2S producing yeast strain Saccharomyces cerevisiae P1Y2 , and the other set was produced using Saccharomyces cerevisiae RC212. Fermentations were conducted in triplicate. At the completion of alcoholic fermentation, wines were pressed and split into three different lees treatments based on settling time (0, 24 and 72 hrs) which were named as heavy, medium and light lees treatment respectively in this progress report. Samples were collected at 0, 2 weeks, and 1, 2, 3, 6, 9 months. Volatile sulfur compounds were analyzed by solid phase microextraction-gas chromatography-pulsed flame photometric detection (SPME-GC-PFPD) method. The results showed that hydrogen sulfide (H2S), methylmercaptan (methanethiol) and methyl thioacetate (sulfur containing ester) were the major sulfur compounds of the concern in the wines. Moreover, the concentration of H2S was directly influenced by the type of yeasts in the first month of storage. Wines made from Saccharomyces cerevisiae P1Y2 generally had lower concentration of H2S than those from Saccharomyces cerevisiae RC212. In addition, lower H2S concentration was observed in light lees load than in medium or heavy lees load treatment from Saccharomyces cerevisiae P1Y2 during the first two weeks of aging, whereas more H2S were generated in higher lees loading samples from Saccharomyces cerevisiae RC212. H2S accumulated with time during early aging, and reached to the maximum at one month, then decreased afterwards regardless of types of lees or the amount of lees loaded. Methanethiol also accumulated during aging, and reached its maximum at 2-3 months, then decreased slowly afterwards.

High level of methyl thioacetate was detected in the experimental wines, wines from Saccharomyces cerevisiae RC212 had substantially higher level of methyl thioacetate than those from Saccharomyces cerevisiae P1Y2 regardless of lees level, and the concentration of methyl thioacetate stayed consistent during barrel aging. Heavy lees generally lead to more dimethyl sulfide (DMS) accumulation. During the aging storage, DMS reached to peak level at 2-3 months, decreased at 6 months, and then continued accumulating at 9 months. The levels of other sulfur compounds (carbon disulfide, dimethyl disulfide and dimethyl trisulfide) were very low for flavor contribution.  The samples from collaborating wineries were also analyzed, but the results were complicated due to various treatments and remedies performed at winery. H2S was the major volatile sulfur compound in those winery samples, especially at the beginning of the barrel aging. Some winery samples also had high levels of methyl thioacetate and methanethiol. Although various remedies were performed in wineries to remove H2S , the levels of methyl thioacetate were very high in many of the wines after 6 to 9 months of aging. High concentration of methanethiol was also observed in many wines. More winery samples were studied for 2014 harvest and high levels of hydrogen sulfide, DMS and methyl thioacetate were detected in most of those wines. We collaborated with Dr. James Osborne further studied the effect of YAN on volatile sulfur formation. Detailed results were submitted separately as a single report. The study showed that the levels of YAN did not affect the generation of MeSH, CS2, DMDS and DMDS. However, the amount of YAN and the type of YAN did affect the formation of thioactates by Saccharomyces cerevisiae UCD522. DAP addition generated higher thioacetates.  We also studied the effect of elemental sulfur on volatile sulfur composition. Although the residual sulfur affected hydrogen sulfide production during fermentation (see the separate report), sulfur addition did not affect the volatile sulfur compounds in the final wine except for methyl thioacetate. Sulfur addition generated more methyl thioacetate by both Saccharomyces cerevisiae P1Y2 and UCD522. High levels of methyl thioacetate could be an important issue for winery. Methyl thioacetate impart sulfur off-flavor in wine, it can also be converted to methanethiol that has a very low sensory threshold. The results suggested the methyl thioacetate and methanethiol could be the major culprits for sulfur off-flavor development during barrel aging. This new finding is significant and needs to have further investigation in future.

Chiral Terpenes – Quantitation, Threshold Determination and Sensory Impact on Aromatic White Wines

The specific accomplishment of the last year for this project was the development of a quantitative method using MDGC to measure chiral terpenes in white wine. Two hundred and three white wine samples, Pinot Gris and Riesling with diverse residual sugars had been already collected from different places of origin all over the world. Wines were donated from top wine companies from New Zealand, New York, Australia, Germany, Oregon, Washington, France, to name a few. Fifteen chiral mono-terpene compounds were collected from head space solid phase micro-extraction coupled with multidimensional GC-MS with stable dilution isotope quantification analysis. Results for the Pinot Gris wines are presented in this report. In short here were difference sin chiral terpene content between the wines. Additional differentiation was established for place of origin of Pinot Gris wines based on chiral terpene content.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

The task we are undertaking is fundamental. There is currently no quantitative method for determining tannin and pigmented tannin. It is not understood to what extent certain compounds contribute to the overall color of aged (> 2 years old) wine, nor the relative abundances of those compounds. Furthermore, there are many compounds that comprise the molecular basis of pigmented tannin whose structures are unknown. This investigation aims at using mass spectrometry as a tool for assessing the extent of color contribution due to these compounds, identifying new compounds, and providing a means of identifying the relative abundances for determining which compounds are most highly associated with quality parameters. Once we better understand the molecular basis of pigmented tannin we can provide tools for winemakers to improve the quality of their product. Pursuant to our goals for this year of the project we have (1a) identified many compounds by mass spectrometry which comprise the wine matrix, and many yet which have not been observed. Our FT-ICR experiment for determination of the relative abundance, depletion and accumulation of particular compounds (1b) will be performed in April with our collaborator Professor Nikolai Kuhnert at his laboratories and the Bruker research laboratories in Bremen Germany. Development of synthetic standards (2abc) is in progress as we are still assessing the appropriate standards to synthesize. We have defined the compound classes which comprise the majority of molecular peaks and will be performing further iterative fragmentation of these compounds to determine their structural characteristics in March with fellow anthocyanin researcher Professor Colin Kay at the University of East Anglia. All in all we are on track to accomplish our objectives and maintain pace for the upcoming year of data analysis and subsequent experimentation.

Extended Maceration

In this study we looked at the effect of extended maceration (up to 8 weeks) with daily pumpovers or submerging the cap (also up to 8 weeks) on the sensory and chemical attributes of the resultant Merlot wines.  Chemical measures of polyphenols and basic wine chemistry, along with Descriptive Analysis (DA) comprised three data sets.  Principal component analysis (PCA) applied to the individual data sets discriminated the wines by treatment, with each of the three PCAs capturing 90 – 97{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the treatment variability with in the first three components.  We also used Temporal Dominance of Sensation (TDS) a relatively new sensory technique to evaluate the time component of mouthfeel and taste attributes.

  • Nine descriptive attributes were significantly different.  Maceration was associated with increases in red fruit, bitterness, astringency, drying, and astringent texture, along with a decrease in pepper spice.
  • Descriptive analysis results showed that 8 week extended maceration, whether by submerging the cap (Su8) or with daily pumpovers (Pu8), had similar sensory profiles. Also, the control wines with no extended maceration but with different cap management methods (Po0, Su0 and Punch down (PD0)) were grouped together as similar in profile.
  • The TDS profiles of Po8 and Su8 were different despite each having a similar chemical and descriptive profile, this shows that TDS can pick out sensory nuances that occur over the time course of the wines residence in the mouth that single point methods, such as DA.
  • For all 9 treatments, astringency became the dominant sensation at approximately the same time point.
  • Po0 and PD0 showed a clear temporal progression when compared to the Su0 treatment.
  • Maceration showed a decrease in anthocyanidin concentration that leveled after two weeks, while tannin concentration continued to increase.  The Su0 treatment had the highest anthocyanin measurements.
  • In general, maceration tended to increase compounds associated with apple aroma and decrease fruit/ floral aromas.
  • Maceration gave similar tannin measurements as submerged cap, but a different sensory profile

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.