Multi-year field evaluation of nicotianamine biofortified bread wheat.

Conventional breeding efforts for iron (Fe) and zinc (Zn) biofortification of bread wheat (Triticum aestivum L.) have been hindered by a lack of genetic variation for these traits and a negative correlation between grain Fe and Zn concentrations and yield. We have employed genetic engineering to constitutively express (CE) the rice (Oryza sativa) nicotianamine synthase 2 (OsNAS2) gene and upregulate biosynthesis of two metal chelators - nicotianamine (NA) and 2'-deoxymugineic acid (DMA) - in bread wheat, resulting in increased Fe and Zn concentrations in wholemeal and white flour. Here we describe multi-location confined field trial (CFT) evaluation of a low-copy transgenic CE-OsNAS2 wheat event (CE-1) over 3 years and demonstrate higher concentrations of NA, DMA, Fe, and Zn in CE-1 wholemeal flour, white flour, and white bread and higher Fe bioavailability in CE-1 white flour relative to a null segregant (NS) control. Multi-environment models of agronomic and grain nutrition traits revealed a negative correlation between grain yield and grain Fe, Zn, and total protein concentrations, yet no correlation between grain yield and grain NA and DMA concentrations. White flour Fe bioavailability was positively correlated with white flour NA concentration, suggesting that NA-chelated Fe should be targeted in wheat Fe biofortification efforts.

The Fungal Microbiome Is an Important Component of Vineyard Ecosystems and Correlates with Regional Distinctiveness of Wine.

The flavors of fermented plant foods and beverages are formed by microorganisms, and in the case of wine, the location and environmental features of the vineyard site also imprint the wine with distinctive aromas and flavors. Microbial growth and metabolism play an integral role in wine production, by influencing grapevine health, wine fermentation, and the flavor, aroma, and quality of finished wines. The contributions by which microbial distribution patterns drive wine metabolites are unclear, and while flavor has been correlated with fungal and bacterial composition for wine, bacterial activity provides fewer sensorially active biochemical conversions than fungi in wine fermentation. Here, we collected samples across six distinct wine-growing areas in southern Australia to investigate regional distribution patterns of fungi and bacteria and the association with wine chemical composition. Results show that both soil and must microbiota distinguish wine-growing regions. We found a relationship between microbial and wine metabolic profiles under different environmental conditions, in particular measures of soil properties and weather. Fungal communities are associated with wine regional distinctiveness. We found that the soil microbiome is a source of grape- and must-associated fungi and suggest that weather and soil could influence wine characteristics via the soil fungal community. Our report describes a comprehensive scenario of wine microbial biogeography where microbial diversity responds to the surrounding environment and correlates with wine composition and regional characteristics. These findings provide perspectives for thoughtful human practices to optimize food composition through understanding fungal activity and abundance.IMPORTANCE The composition of soil has long been thought to provide wine with characteristic regional flavors. Here, we show that for vineyards in southern Australia, the soil fungal communities are of primary importance for the aromas found in wines. We propose a mechanism by which fungi can move from the soil through the vine.

Statistical modelling coupled with LC-MS analysis to predict human upper intestinal absorption of phytochemical mixtures.

A diet rich in phytochemicals confers benefits for health by reducing the risk of chronic diseases via regulation of oxidative stress and inflammation (OSI). For optimal protective bio-efficacy, the time required for phytochemicals and their metabolites to reach maximal plasma concentrations (Tmax) should be synchronised with the time of increased OSI. A statistical model has been reported to predict Tmax of individual phytochemicals based on molecular mass and lipophilicity. We report the application of the model for predicting the absorption profile of an uncharacterised phytochemical mixture, herein referred to as the 'functional fingerprint'. First, chemical profiles of phytochemical extracts were acquired using liquid chromatography mass spectrometry (LC-MS), then the molecular features for respective components were used to predict their plasma absorption maximum, based on molecular mass and lipophilicity. This method of 'functional fingerprinting' of plant extracts represents a novel tool for understanding and optimising the health efficacy of plant extracts.

Soluble Protein and Amino Acid Content Affects the Foam Quality of Sparkling Wine.

Proteins and amino acids are known to influence the foam characteristics of sparkling wines. However, it is unclear to what extent they promote foam formation and/or stability. This study aimed to investigate the effect of protein content and amino acid composition, measured via the bicinchoninic acid assay and high-performance liquid chromatography, respectively, on the foaming properties of 28 sparkling white wines, made by different production methods. Foam volume and stability were determined using a robotic pourer and computer vision algorithms. Modifications were applied to the protein determination method involving the use of yeast invertase as a standard in order to improve quantification accuracy. The protein content was found to be significantly correlated to parameters representative of foam stability, as were the amino acids arginine, asparagine, histidine, and tyrosine. Additionally, the production method was found to influence the foam collar height, which favored foaming in Méthode Traditionnelle wines over other those made by production methods. Understanding the contributions of key wine constituents to the visual and mouthfeel parameters of sparkling wine will enable more efficient production of high-quality wines.

Dietary Phytochemicals Promote Health by Enhancing Antioxidant Defence in a Pig Model.

Phytochemical-rich diets are protective against chronic diseases and mediate their protective effect by regulation of oxidative stress (OS). However, it is proposed that under some circumstances, phytochemicals can promote production of reactive oxygen species (ROS) in vitro, which might drive OS-mediated signalling. Here, we investigated the effects of administering single doses of extracts of red cabbage and grape skin to pigs. Blood samples taken at baseline and 30 min intervals for 4 hours following intake were analyzed by measures of antioxidant status in plasma, including Trolox equivalent antioxidant capacity (TEAC) and glutathione peroxidase (GPx) activity. In addition, dose-dependent production of hydrogen peroxide (H2O2) by the same extracts was measured in untreated commercial pig plasma in vitro. Plasma from treated pigs showed extract dose-dependent increases in non-enzymatic (plasma TEAC) and enzymatic (GPx) antioxidant capacities. Similarly, extract dose-dependent increases in H2O2 were observed in commercial pig plasma in vitro. The antioxidant responses to extracts by treated pigs were highly correlated with their respective yields of H2O2 production in vitro. These results support that dietary phytochemicals regulate OS via direct and indirect antioxidant mechanisms. The latter may be attributed to the ability to produce H2O2 and to thereby stimulate cellular antioxidant defence systems.

Influence of Production Method on the Chemical Composition, Foaming Properties, and Quality of Australian Carbonated and Sparkling White Wines.

The chemical composition (protein, polysaccharide, amino acid, and fatty acid/ethyl ester content), foaming properties, and quality of 50 Australian sparkling white wines, representing the four key production methods, that is, Méthode Traditionelle (n = 20), transfer (n = 10), Charmat (n = 10), and carbonation (n = 10), were studied. Méthode Traditionelle wines were typically rated highest in quality and were higher in alcohol and protein contents, but lower in residual sugar and total phenolics, than other sparkling wines. They also exhibited higher foam volume and stability, which might be attributable to higher protein concentrations. Bottle-fermented Méthode Traditionelle and transfer wines contained greater proportions of yeast-derived mannoproteins, whereas Charmat and carbonated wines were higher in grape-derived rhamnogalacturonans; however, total polysaccharide concentrations were not significantly different between sparkling wine styles. Free amino acids were most abundant in carbonated wines, which likely reflects production via primary fermentation only and/or the inclusion of nontraditional grape varieties. Fatty acids and their esters were not correlated with foaming properties, but octanoic and decanoic acids and their ethyl esters were present in Charmat and carbonated wines at significantly higher concentrations than in bottle-fermented wines and were negatively correlated with quality ratings. Research findings provide industry with a better understanding of the compositional factors driving the style and quality of sparkling white wine.

Drosophila-associated yeast species in vineyard ecosystems.

Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem.

Variation in 4-mercapto-4-methyl-pentan-2-one release by Saccharomyces cerevisiae commercial wine strains.

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.

Wild Saccharomyces cerevisiae strains display biofilm-like morphology in contact with polyphenols from grapes and wine.

Polyphenols are a major component of wine grapes, and contribute to color and flavor, but their influence upon yeast growth forms has not been investigated. In this work we have studied the effect of polyphenols on the ability of natural isolates of wine-related Saccharomyces cerevisiae strains to form biofilms attaching to plastic surfaces, to grow as mat colonies, to invade media, and to display filamentous growth. The use of carbon- and nitrogen-rich or deficient media simulated grape juice fermentation conditions. The addition of wine polyphenols to these media affected biofilm formation, and cells exhibited a wide variety of invasiveness and mat formation ability with associated different growth and footprint patterns. Microscopic observation revealed that some strains switched to filamentous phenotypes which were able to invade media. The wide range of phenotypic expression observed could have a role in selection of strains suitable for inoculated wine fermentations and may explain the persistence of yeast strains in vineyard and winery environments.

Biodiversity, dynamics and ecology of bacterial community during grape marc storage for the production of grappa.

The Italian spirit obtained from grape marc, grappa, is produced by an extended storage of the marc which allows alcoholic fermentation. Bacterial populations can develop and are associated with off-flavour production. Grape marc acidification before storage is a common practice in distilleries to control bacterial proliferation. Few studies have been published on the microbial biodiversity in grape marc and no information exists about microbiology of acidified marcs and physiological properties needed for colonizing such an environment. The aim of this study was to investigate the composition and dynamics of grape marc bacterial populations during the long-period storage by microbiological analyses of acidified and untreated marcs. Eight bacterial species were identified by ARDRA - 16s rRNA sequencing at the beginning of the fermentation. Among them the bacterial species of Tatumella terrea, Acetobacter ghanensis and Tatumella ptyseos were identified for the first time in a wine environment. In later stages Oenococcus oeni and members of the Lactobacillus plantarum group became dominant in acidified and non-acidified grape marc, respectively. Further molecular typing of L. plantarum isolates yielded 39 strains. To explain the prevalence of L. plantarum in untreated samples, all strains were tested for potential antimicrobial activity and for biofilm formation ability. Although no antimicrobial activity was found, many strains exhibited the ability to form a biofilm, which may confer an ecological advantage to these strains and their dominance during marc storage.

Protection of C. elegans from anoxia by HYL-2 ceramide synthase.

Oxygen deprivation is rapidly deleterious for most organisms. However, Caenorhabditis elegans has developed the ability to survive anoxia for at least 48 hours. Mutations in the DAF-2/DAF-16 insulin-like signaling pathway promote such survival. We describe a pathway involving the HYL-2 ceramide synthase that acts independently of DAF-2. Loss of the ceramide synthase gene hyl-2 results in increased sensitivity of C. elegans to anoxia. C. elegans has two ceramide synthases, hyl-1 and hyl-2, that participate in ceramide biogenesis and affect its ability to survive anoxic conditions. In contrast to hyl-2(lf) mutants, hyl-1(lf) mutants are more resistant to anoxia than normal animals. HYL-1 and HYL-2 have complementary specificities for fatty acyl chains. These data indicate that specific ceramides produced by HYL-2 confer resistance to anoxia.

Identifying key residues of sphinganine-1-phosphate lyase for function in vivo and in vitro.

Sphinganine-1-phosphate lyase (Dpl1p) is a highly conserved enzyme of sphingolipid metabolism that catalyzes the irreversible degradation of sphingoid base phosphates, which are potent signaling molecules. Sphingoid base phosphates play a vital role in cell survival, proliferation, migration, heat stress, and cell wall integrity pathways. Little is known about the structure and regulation of Dpl1p. In this study, we have undertaken a combined computational modeling and mutagenesis approach for structure-function analysis of Dpl1p to discover possible modes of regulation. Our results identify important residues for catalysis in Dpl1p and confirm it as an integral endoplasmic reticulum-resident protein. Results further indicate that Dpl1p is most likely not regulated spatially. Importantly, we demonstrate that Dpl1p exists as an oligomer and that polar residues in its transmembrane domain are required for its full function in vivo but not for its localization or for its catalytic activity in vitro.

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation.

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.

Genetic determinants of volatile-thiol release by Saccharomyces cerevisiae during wine fermentation.

Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.