Glycosidically-Bound Volatile Phenols Linked to Smoke Taint: Stability during Fermentation with Different Yeasts and in Finished Wine.

When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing ß-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p-cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-ß-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae ß-glucosidase activity appear to be influenced by the VP moiety.

The Lavender Fertilized Anterior Cruciate Ligament Reconstruction: A Quadriceps Tendon All-Inside Reconstruction Fertilized With Bone Marrow Concentrate, Demineralized Bone Matrix, and Autograft Bone.

The use of biologics in sports medicine is increasing rapidly. Bone marrow concentrate has recently increased in popularity because it includes mesenchymal stem cells which, combined with AlloSync Pure, could lead to better incorporation and healing. The mixture of bone marrow concentrate and Allosync Pure can be used in anterior cruciate ligament reconstruction. We recently expanded on this approach with the addition of saving the host bone normally lost from tunnel reaming, using the GraftNet. After harvesting the autograft bone, we combine it with the AlloSync Pure and bone marrow concentrate. In this Technical Note, we show how this unique biologic composite is obtained and then added back into the tunnels on both the femur and tibia during a quadriceps tendon autograft all-inside anterior cruciate ligament reconstruction.

Smoke from simulated forest fire alters secondary metabolites in Vitis vinifera L. berries and wine.

MAIN CONCLUSION: The exposure of Vitis vinifera L. berries to forest fire smoke changes the concentration of phenylpropanoid metabolites in berries and the resulting wine. The exposure of Vitis vinifera L. berries (i.e., wine grapes) to forest fire smoke can lead to a wine defect known as smoke taint that is characterized by unpleasant "smoky" and "ashy" aromas and flavors. The intensity of smoke taint is associated with the concentration of organoleptic volatile phenols that are produced during the combustion-mediated oxidation of lignocellulosic biomass and subsequently concentrated in berries prior to fermentation. However, these same smoke-derived volatile phenols are also produced via metabolic pathways endogenous to berries. It follows then that an influx of exogenous volatile phenols (i.e., from forest fire smoke) could alter endogenous metabolism associated with volatile phenol synthesis, which occurs via the shikimic acid/phenylpropanoid pathways. The presence of ozone and karrikins in forest fire smoke, as well as changes to stomatal conductance that can occur from exposure to forest fire smoke also have the potential to influence phenylpropanoid metabolism. This study demonstrated changes in phenylpropanoid metabolites in Pinot noir berries and wine from three vineyards following the exposure of Vitis vinifera L. vines to simulated forest fire smoke. This included changes to metabolites associated with mouth feel and color in wine, both of which are important sensorial qualities to wine producers and consumers. The results reported are critical to understanding the chemical changes associated with smoke taint beyond volatile phenols, which in turn, may aid the development of preventative and remedial strategies.

Detailed characterization of glycosylated sensory-active volatile phenols in smoke-exposed grapes and wine.

The exposure of Vitis vinifera L. vines to smoke from wildland fires can alter the chemical composition of the berries, such that the resulting wine can possess a defect known as smoke-taint. This work constitutes a complete method for the analysis of simple volatile phenol glycosides (VP-glycosides) that can be elevated in berries and wine following smoke exposure. We synthesized 16 model VP-glycosides, four of which are not reported previously, to facilitate method development. Fragmentation analysis using high-resolution accurate-mass spectrometry demonstrated that the glycone and aglycone influenced the fragmentation pattern of VP-glycosides. Diagnostic fragmentation patterns for the synthesized VP-glycosides were applied to identify several VP-glycosides in smoke-exposed berries and wine. The fragmentation pattern of VP-disaccharides should facilitate the characterization of modified glycones. Putative non-VP glycosides elevated in smoke-exposed berries are demonstrated for the first time. In tandem with VP-glycosides, such compounds may contribute to the expression of smoke taint.

Fermentation-Guided Natural Products Isolation of a Grape Berry Triacylglyceride that Enhances Ethyl Ester Production.

A full understanding of the origin, formation and degradation of volatile compounds that contribute to wine aroma is required before wine style can be effectively managed. Fractionation of grapes represents a convenient and robust method to simplify the grape matrix to enhance our understanding of the grape contribution to volatile compound production during yeast fermentation. In this study, acetone extracts of both Riesling and Cabernet Sauvignon grape berries were fractionated and model wines produced by spiking aliquots of these grape fractions into model grape juice must and fermented. Non-targeted SPME-GCMS analyses of the wines showed that several medium chain fatty acid ethyl esters were more abundant in wines made by fermenting model musts spiked with certain fractions. Further fractionation of the non-polar fractions and fermentation of model must after addition of these fractions led to the identification of a mixture of polyunsaturated triacylglycerides that, when added to fermenting model must, increase the concentration of medium chain fatty acid ethyl esters in wines. Dosage-response fermentation studies with commercially-available trilinolein revealed that the concentration of medium chain fatty acid ethyl esters can be increased by the addition of this triacylglyceride to model musts. This work suggests that grape triacylglycerides can enhance the production of fermentation-derived ethyl esters and show that this fractionation method is effective in segregating precursors or factors involved in altering the concentration of fermentation volatiles.

Quantitating Volatile Phenols in Cabernet Franc Berries and Wine after On-Vine Exposure to Smoke from a Simulated Forest Fire.

Smoke-taint is a wine defect linked to organoleptic volatile phenols (VPs) in Vitis vinifera L. berries that have been exposed to smoke from wildland fires. Herein, the levels of smoke-taint-associated VPs are reported in Cabernet Franc berries from veraison to commercial maturity and in wine after primary fermentation following on-vine exposure to simulated wildland fire smoke. VPs increased after smoke exposure were rapidly stored as acid-labile conjugates, and the levels of both free VPs and conjugated forms remained constant through ripening to commercial maturity. An increase in total VPs after primary fermentation suggested the existence of VP-conjugates other than the acid-labile VP-glycosides already reported. This conclusion was supported with base hydrolysis on the same samples. Relative to published results, the data suggested a multifactorial regional identity for smoke-taint and they inform efforts to produce a predictive model for perceptible smoke-taint in wine based on the chemical composition of smoke-exposed berries.

Quantitating Organoleptic Volatile Phenols in Smoke-Exposed Vitis vinifera Berries.

Accurate methods for quantitating volatile phenols (i.e., guaiacol, syringol, 4-ethylphenol, etc.) in smoke-exposed Vitis vinifera berries prior to fermentation are needed to predict the likelihood of perceptible smoke taint following vinification. Reported here is a complete, cross-validated analytical workflow to accurately quantitate free and glycosidically bound volatile phenols in smoke-exposed berries using liquid-liquid extraction, acid-mediated hydrolysis, and gas chromatography-tandem mass spectrometry. The reported workflow addresses critical gaps in existing methods for volatile phenols that impact quantitative accuracy, most notably the effect of injection port temperature and the variability in acid-mediated hydrolytic procedures currently used. Addressing these deficiencies will help the wine industry make accurate, informed decisions when producing wines from smoke-exposed berries.

Potential grape-derived contributions to volatile ester concentrations in wine.

Grape composition affects wine flavour and aroma not only through varietal compounds, but also by influencing the production of volatile compounds by yeast. C9 and C12 compounds that potentially influence ethyl ester synthesis during fermentation were studied using a model grape juice medium. It was shown that the addition of free fatty acids, their methyl esters or acyl-carnitine and acyl-amino acid conjugates can increase ethyl ester production in fermentations. The stimulation of ethyl ester production above that of the control was apparent when lower concentrations of the C9 compounds were added to the model musts compared to the C12 compounds. Four amino acids, which are involved in CoA biosynthesis, were also added to model grape juice medium in the absence of pantothenate to test their ability to influence ethyl and acetate ester production. ß-Alanine was the only one shown to increase the production of ethyl esters, free fatty acids and acetate esters. The addition of 1 mg∙L(-1) ß-alanine was enough to stimulate production of these compounds and addition of up to 100 mg∙L(-1) ß-alanine had no greater effect. The endogenous concentrations of ß-alanine in fifty Cabernet Sauvignon grape samples exceeded the 1 mg∙L(-1) required for the stimulatory effect on ethyl and acetate ester production observed in this study.

A methyltransferase essential for the methoxypyrazine-derived flavour of wine.

Methoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F2 progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3-isobutyl-2-methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.

A novel tool for studying auxin-metabolism: the inhibition of grapevine indole-3-acetic acid-amido synthetases by a reaction intermediate analogue.

An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA) by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP) mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with K(i)-values 17-68-fold lower than the respective K(m)-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5-20 µM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function.

Grape contribution to wine aroma: production of hexyl acetate, octyl acetate, and benzyl acetate during yeast fermentation is dependent upon precursors in the must.

Wine is a complex consumer product produced predominately by the action of yeast upon grape juice musts. Model must systems have proven ideal for studies of the effects of fermentation conditions on the production of certain wine volatiles. To identify grape-derived precursors to acetate esters, model fermentation systems were developed by spiking precursors into model must at different concentrations. Solid-phase microextraction-gas chromatgraphy mass spectrometry analysis of the fermented wines showed that a variety of grape-derived aliphatic alcohols and aldehydes are precursors to acetate esters. The C6 compounds hexan-1-ol, hexenal, (E)-2-hexen-1-ol, and (E)-2-hexenal are all precursors to hexyl acetate, and octanol and benzyl alcohol are precursors to octyl acetate and benzyl acetate, respectively. In these cases, the postfermentation concentration of an acetate ester increased proportionally with the prefermentation concentration of the respective precursor in the model must. Determining viticultural or winemaking methods to alter the prefermentation concentration of precursor compounds or change the precursor-to-acetate ester ratio will have implications upon the final flavor and aroma of wines.

Two O-methyltransferases involved in the biosynthesis of methoxypyrazines: grape-derived aroma compounds important to wine flavour.

Methoxypyrazines (MPs) are volatile, grape-derived aroma compounds that contribute to the distinct herbaceous characters of some wines. Although the full pathway leading to MP production has not been elucidated, there is strong evidence that the final step involves the methylation of non-volatile hydroxypyrazine (HP) precursors. Two cDNA encoding O-methyltransferases (OMTs) that have homology to an enzyme previously purified and shown to catalyse the methylation of HPs were isolated from Cabernet Sauvignon. Recombinant protein from the cDNAs (VvOMT1 and VvOMT2) was produced in E. coli and activity assays demonstrated that both encode OMTs able to methylate HPs to produce MPs, however both showed greatest activity against the flavonol quercetin. VvOMT1 has higher catalytic activity against isobutyl hydroxypyrazine compared to isopropyl hydroxypyrazine, whereas the converse is true for VvOMT2. The timing of the expression of VvOMT1 in the skin and the flesh of developing Cabernet Sauvignon grape berries was associated with the period of MP accumulation in these tissues, while VvOMT2 expression was greatest in roots, which were found to contain high levels of MPs. The MP composition of these tissues also reflects the relative levels of expression of these genes and their substrate preference. The identification of genes responsible for MP production in grapevine will help in understanding the effect of different viticultural and environmental factors on MP accumulation.