The Varietal Influence of Flavour Precursors from Grape Marc on Monoterpene and C13-Norisoprenoid Profiles in Wine as Determined by Membrane-Assisted Solvent Extraction (MASE) GC-MS.

The winemaking by-product grape marc (syn. pomace) contains significant quantities of latent flavour in the form of flavour precursors which can be extracted and used to modulate the volatile composition of wine via chemical hydrolysis. Varietal differences in grapes are widely known with respect to their monoterpene content, and this work aimed to extend this knowledge into differences due to cultivar in volatiles derived from marc precursors following wine-like storage conditions. Marc extracts were produced from floral and non-floral grape lots on a laboratory-scale and from Muscat Gordo Blanco marc on a winery -scale, added to a base white wine for storage over five to six months, before being assessed using a newly developed membrane-assisted solvent extraction gas chromatography-mass spectrometry (GC-MS) method. The geraniol glucoside content of the marc extracts was higher than that of juices produced from each grape lot. In all wines with added marc extract from a floral variety, geraniol glucoside concentration increased by around 150-200%, with increases also observed for non-floral varieties. The relative volatile profile from extracts of the floral varieties was similar but had varied absolute concentrations. In summary, while varietally pure extracts would provide the greatest control over flavour outcomes when used in winemaking, aggregated marc parcels from floral cultivars may provide a mechanism to simplify the production logistics of latent flavour extracts for use in the wine sector.

Effects of Feeding either Red or White Grape Marc on Milk Production and Methane Emissions from Early-Lactation Dairy Cows.

Globally, annual production of grape marc (GM), the residue of skins, seeds and stems remaining after making wine, has been estimated to be approximately nine million tonnes. No previous studies have compared effects on milk production and methane emissions when GM from either red or white grapes was fed to dairy cows. This experiment examines the effects of partial replacement of a perennial ryegrass (Lolium perenne L.) based diet with GM from either red or white grapes on yield and composition of milk and methane emissions. Thirty-two Holstein dairy cows in early lactation were offered either a control diet containing 15.0 kg dry matter (DM) of freshly harvested perennial ryegrass and 5.2 kg of a concentrate mix, or a diet similar to the control diet but with 5 kg DM of ryegrass replaced with 5 kg DM of GM from red grapes (RGM), or a diet similar to the RGM diet except the GM was from white grapes (WGM). Individual cow feed intakes, milk yields, and methane emissions were measured. Both diets containing GM decreased milk yields by approximately 10% and methane emissions by 15%. When fed to dairy cows, GM reduces methane emissions but at the cost of decreased milk production.

Understanding Yeast Impact on 1,1,6-Trimethyl-1,2-dihydronaphthalene Formation in Riesling Wine through a Formation-Pathway-Informed Hydrolytic Assay.

The occurrence in Riesling wine of the potent odorant 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) is dependent upon vineyard and winemaking conditions, and TDN can have a prominent impact on the attributes of a wine after years in a bottle. As such, immediately assessing the impact of vineyard or winery treatments on future TDN formation requires forced creation of the aroma compound under non-wine-like conditions from other precursors. Here, we use a Box-Behnken approach and known TDN end points in commercial wines to optimize the conditions (pH, temperature, and time) of a "total TDN" hydrolytic assay for Riesling wine, which was intended to not interfere with yeast-derived formation pathways. The new assay (75 °C, pH 1.7, and 60 min) was used to determine the role of industry-relevant commercial yeasts as well as novel hybrid yeast strains on total TDN concentrations in young Riesling wines. While significant differences were observed between some yeasts, the impact of defoliation as a viticultural intervention outweighed yeast effects, suggesting that elevated TDN concentrations in wine are likely due to grape growing conditions and cannot be readily reduced or compensated for in the winery.

Exploiting Compositionally Similar Grape Marc Samples to Achieve Gradients of Condensed Tannin and Fatty Acids for Modulating In Vitro Methanogenesis.

Ruminants produce large amounts of the greenhouse gas, methane, which can be reduced by supplementing feed with products that contain anti-methanogenic compounds, such as the solid winemaking by-product, grape marc. The aim of this study was to exploit compositional differences in grape marc to better understand the roles of condensed tannin and fatty acids in altering methanogenesis in a ruminant system. Grape marc samples varying in tannin extractability, tannin size and subunit composition, and fatty acid or tannin concentrations were selected and incubated in rumen fluid using an in vitro batch fermentation approach with a concentrate-based control. Four distinct experiments were designed to investigate the effects on overall fermentation and methane production. Generally, fatty acid concentration in grape marc was associated with decreased total gas volumes and volatile fatty acid concentration, whereas increased condensed tannin concentration tended to decrease methane percentage. Smaller, extractable tannin was more effective at reducing methane production, without decreasing overall gas production. In conclusion, fatty acids and tannin concentration, and tannin structure in grape marc play a significant role in the anti-methanogenic effect of this by-product when studied in vitro. These results should be considered when developing strategies to reduce methane in ruminants by feeding grape marc.

Hydroxycinnamoyl Glucose and Tartrate Esters and Their Role in the Formation of Ethylphenols in Wine.

Synthesized p-coumaroyl and feruloyl l-tartrate esters were submitted to Brettanomyces bruxellensis strains AWRI 1499, AWRI 1608, and AWRI 1613 to assess their role as precursors to ethylphenols in wine. No evolution of ethylphenols was observed. Additionally, p-coumaroyl and feruloyl glucose were synthesized and submitted to B. bruxellensis AWRI 1499, which yielded both 4-ethylphenol and 4-ethylguaiacol. Unexpected chemical transformations of the hydroxycinnamoyl glucose esters during preparation were investigated to prevent these in subsequent synthetic attempts. Photoisomerization gave an isomeric mixture containing the trans-esters and undesired cis-esters, and acyl migration resulted in a mixture of the desired 1-O-ß-ester and two additional migrated forms, the 2-O-α- and 6-O-α-esters. Theoretical studies indicated that the photoisomerization was facilitated by deprotonation of the phenol, and acyl migration is favored during acidic, nonaqueous handling. Preliminary LC-MS/MS studies observed the migrated hydroxycinnamoyl glucose esters in wine and allowed for identification of feruloyl glucose in red wine for the first time.

Survey of the Variation in Grape Marc Condensed Tannin Composition and Concentration and Analysis of Key Compositional Factors.

Grape marc contains a number of compounds with potential antimethanogenic activity in ruminants, including condensed tannins (CTs). Using direct phloroglucinolysis, a survey of CT chemistry across 66 grape marc samples showed diversity in concentration (6.9 to 138.8 g/kg of dry matter). Concentration was found to be independent of CT composition, although all compositional variables were significantly correlated (P < 0.0001). Twenty samples diverse in CT were selected from a cluster analysis and analyzed for compounds relevant to ruminant digestion and methanogenesis, including metabolizable energy (6.6-12.0 MJ/kg DM), crude protein (3.2-14.4% DM), neutral detergent fiber (18.4-61.4% DM), and ethanol soluble carbohydrates (2.0-40.6% DM). Fatty acid concentrations varied throughout the 20 samples (5.2-184.5 g/kg DM), although fatty acid profile showed two distinct groups. Grape marc varies widely in nutritional value, and in compounds that have been linked with changes in ruminant digestion and methane emissions.

Evaluation of direct phloroglucinolysis and colorimetric depolymerization assays and their applicability for determining condensed tannins in grape marc.

To determine the optimum methods for determining condensed tannin (CT) content in grape marc, butanol-hydrochloric acid assays and phloroglucinolysis were adapted for use, applied to a range of grape marc types, and the methods compared. Porter's assay (butanol-HCl) was found to give unreliable results due to nonlinear color responses to grape skin and seed tannin concentrations, whereas the modification to include acetone (Grabber's assay) overcame this. Differences between skin and seed tannin responses highlighted the need to adequately select the correct grape tannin standard, and the formation of pH-dependent color was accounted for through acidification of blank samples. For phloroglucinolysis, the inability to remove highly bound tannins from cell wall material was highlighted, although a measure of tannins remaining post-phloroglucinolysis (Grabber's assay) showed a trend with the level of exposure to oxidative storage or processing conditions. The comparison of CT concentrations from phloroglucinolysis and Grabber's assay gave poor correlation coefficients.

Di-tert-butyl (2R,3R)-2-{[(2E)-3-(4-acet-yloxy-3-meth-oxy-phen-yl)prop-2-eno-yl]-oxy}-3-hy-droxy-butane-dioate.

In the title mol-ecule, C(24)H(32)O(10), one tert-butyl ester group is folded towards the central benzene ring while the other is directed away. The acetyl group is almost perpendicular to the benzene ring to which it is connected [C-C-O-C torsion angle = 90.4 (12)°]. The conformation about the ethene bond [1.313 (7) Å] is E. The atoms of the benzene ring and its attached ester group and part of the hy-droxy tert-butyl ester side chain are disordered over two sets of sites in a 50:50 ratio. Linear supra-molecular chains along the a axis mediated by hy-droxy-carbonyl O-H⋯O hydrogen bonds feature in the crystal packing. The same H atom also partakes in an intra-molecular O-H⋯O inter-action.

Hydroxycinnamic acid ethyl esters as precursors to ethylphenols in wine.

A method for determining ethyl coumarate and ethyl ferulate in wine using GC-MS with deuterium-labeled analogues has been developed and used to measure the evolution of these two esters during the production of two commercial monovarietal red wines, cv. Grenache and Shiraz. During fermentation, the concentration of ethyl coumarate rose from low levels to 0.4 mg/L in Grenache and 1.6 mg/L in Shiraz wines. These concentrations then increased further during barrel aging to 1.4 and 3.6 mg/L, respectively. The concentration of ethyl ferulate was much lower, reaching a maximum of only 0.09 mg/L. Conversion of ethyl coumarate and ethyl ferulate to their corresponding ethylphenols was observed during fermentations of a synthetic medium with two strains of Dekkera bruxellensis (AWRI 1499 and AWRI 1608), while a third (strain AWRI 1613) produced no ethylphenols at all from these precursors. Strains AWRI 1499 and 1608 produced 4-ethylphenol from ethyl coumarate in 68% and 57% yields, respectively. The corresponding yields of 4-ethylguaiacol from ethyl ferulate were much lower, 7% and 3%. Monitoring of ethyl coumarate and ethyl ferulate concentration during the Dekkera fermentations showed that the selectivity for ethylphenol production according to yeast strain and the precursor was principally a result of variation in esterase activity. Consequently, ethyl coumarate can be considered to be a significant precursor to 4-ethylphenol in wines affected by these two strains of Brettanomyces/Dekkera yeast, while ethyl ferulate is not an important precursor to 4-ethylguaiacol.

1,4-Di-tert-butyl (2R,3R)-2-({(2E)-3-[4-(acet-yloxy)phen-yl]prop-2-eno-yl}-oxy)-3-hy-droxy-butane-dioate.

The title compound, C(23)H(30)O(9), has an approximate T-shape with the tert-butyl ester groups lying either side of the benzene ring. The acetyl group is almost perpendicular to the benzene ring to which it is connected [C-C-O-C torsion angle = -106.7 (3)°]. The conformation about the C=C double bond [1.331 (4) Å] is E. Linear supra-molecular chains along the a axis mediated by hy-droxy-carbonyl O-H⋯O hydrogen bonds feature in the crystal packing. The same H atom is also involved in an intra-molecular O-H⋯O inter-action.