Assessments of Difficult to Ferment Juices

The major goal of this project is to uncover the causes of chronically difficult to ferment juices. These juices are defined as those not due to fermentation management failures and inattention to nutritional needs of the yeast and maintenance of permissive growth and fermentation conditions. These juices are often derived from the same vineyard or block of a vineyard, and other similarly managed vineyards and blocks display normal fermentation kinetics. One class of these difficult to ferment juices is characterized by a high proline to arginine ratio. We have confirmed in several yeast strains that mannitol accumulates within the yeast in these juices and that this is associated with the presence of oxidative stress. To date all of the commercial strains tested are sensitive to these juices and reduce fermentation capacity. We have also confirmed the inhibitory role of previously identified lactic acid bacteria in yeast fermentation but have 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 progeny cells 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 the need for continued induction. We have identified several other genera of lactic acid bacteria as well as acetic acid bacteria from arrested wines that are also capable of inducing the [GAR+] prion in wine yeast. We have received samples from over 53 wineries that have suspected bacterial inhibition of fermentation and have been able to isolate [GAR+] pion-inducing bacteria from many of these wines. Further we have shown that bacteria isolated from stuck wines, when grown in growth-permissive media then removed from the medium via filtration the inducer is still present in the filtrate and capable of  inducing the prion in wine strains of S. cerevisiae. Thus the inducer may be present in the absence of viable bacteria if the fermentation had bacteria present at some point. Controlling the presence of these bacteria is therefore important under production conditions.

Characterization of Aroma Volatiles and their Glycosidic Precursors in Grapes and Wines

The complex aroma of wine is derived from many sources, with grape-derived components being responsible for the varietal character. The ability to monitor grape aroma compounds would allow for better understanding of how vineyard practices and winemaking processes influence the final volatile composition of the wine. Previously we developed a procedure using GC-MS combined with solid-phase microextraction (SPME) for profiling the free volatile compounds in grapes and wines. We have also recently developed a method for monitoring the ‘aroma potential’ of grapes and wines without the need for initial isolation of the glycoside precursor fraction. However, this method still depends on indirect measurement of the glycosides and acid or enzymatic hydrolysis is needed to release the volatile aglycone which can result in artefact formation. In the current project we validated a novel method using UHPLC-qTOF MS/MS for direct analysis of intact aroma glycosides in grapes with minimal artifactual changes in composition. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for the first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. Finally, we observed that depending on the glycoside monitored, there is differential accumulation of monoterpene glycosides during maturation of Muscat of Alexandria berries. This work sheds important insight into possible biochemical changes in glycosylation during grape berry maturation. In addition, this research will allow us to better understand the effects of viticultural and winemaking practices on grape and wine components that affect flavor.

Tannin Structure-Activity Relation to Red Wine Astringency

The objectives of this proposal have been to do the following:

In a companion project being submitted to the Agricultural Research Institute (California State University research initiative)

II. Conduct extended sensory studies with the University of California, Davis, on a subset of the above wines to determine the relationship between sensory and instrumental analysis of red wine mouthfeel.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.

The overall purpose of this proposal is to determine the role of grape and wine production practices on tannin structure and perception.  In combination, tannin activity will be monitored by high performance liquid chromatography (HPLC) so that a comparison can be made between human perception and HPLC measurement.

Following the second year of activities, excellent progress has been made.  The major activity during the second year of this study has been to monitor the development of berries in approximately 75 Napa Valley blocks.  Monitoring the tannin activity in these blocks is expected to provide information on the role that grape production practices can have in overall grape and corresponding wine tannin.

Red Wine Tannin Interaction with Polysaccharides

The objectives of this proposal are to do the following:

I. Determine variation in tannin activity as a function of polysaccharides structure from yeast and/or grape in red wine.

II. Relate the polysaccharide-tannin interaction variation to the wine mouthfeel.

In a companion project funded by the Agricultural Research Institute (California State University research initiative)

III. Improve the understanding of tannin-polysaccharide interactions consequences on red wine stability.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.
The overall purpose of this proposal is to determine the role of polysaccharides from yeast and/or grape in finished red wine on tannin structure and activity. In combination, competitive interactions between tannins and proteins or polysaccharides will be elucidated by spectrophotometry after variation of matrix parameters, so that an elaboration of a functioning interactions model can be made.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

Seven vintages of UC Davis Oakville Cabernet Sauvignon spanning 35 years was selected for analysis by a complimentary suite of mass spectrometric techniques. The vintages from 1974 to 2009 selected by informal sensory evaluation and basic wine chemistry to be of a representative vertical style were: 1974, 1981, 1988, 1994, 2001, 2003 and 2009. All wines were produced at UC Davis for research purposes and stored in the UC Davis cellar together. This sample was determined to be the most controlled and uniform sample of wines varying by vintage only as could be obtained for the project. Al basic wine chemistry was obtained, pH, ethanol concentration etc. as were various assays for comparison to mass spectrometric data.

The following objective were submitted for the grant proposal under which this work was peformed.

1) Observe the evolution of pigmented tannin throughout aging

a. Employ our method of complimentary mass spectrometric techniques (ICR, QTOF, QTrap) for comprehensive identification of wine matrix compounds.

b. Observe the changes in relative abundance, depletion and accumulation in pigmented tannin composition (35 Year Vertical)

c. Correlate pigmented tannin structural analysis with well-known molecular characteristics (mDP, mass recovery) and newly developed sensory representative analyses (“grippiness”) to better convey the impact of these discoveries

2) Apply existing synthetic technologies.

a. Development of a library of standards for quantitation and calibration.

b. Postulate wine pigment precursors for examination of mechanistic pathways.

c. Employ the standards to quantitate the classes of polymeric pigment in wine.

Wine Research Assays

Phloroglucinolysis was performed on the wines used for the study (Table 1) as well as their extracts (Table 2). The mass recovery demonstrates typical value dropping off well below 80%after 7 years. Molecular mass after 50%elution also follows expected patterns of increase with age. This indicates overall larger tannin in the older wines as supported by McRae et al 2012. The molecular mass is a measure of intact tannins, as opposed to the mDP which measures only those tannins which could be cleaved by phloroglucinol. The inconsistency between these two values is reconciled by the mass recovery, indicating that more complexity of the tannin structure is a factor in the older wines, for instance additional interflavan bonds preventing phloroglucinol attack.

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%v/v aqueous methanol and then tannins were eluted with 66%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.