Fully Automated Trimethylsilyl (TMS) Derivatisation Protocol for Metabolite Profiling by GC-MS

Gas Chromatography-Mass Spectrometry (GC-MS) has long been used for metabolite profiling of a wide range of biological samples. Many derivatisation protocols are already available and among these, trimethylsilyl (TMS) derivatisation is one of the most widely used in metabolomics. However, most TMS methods rely on off-line derivatisation prior to GC-MS analysis. In the case of manual off-line TMS derivatisation, the derivative created is unstable, so reduction in recoveries occurs over time. Thus, derivatisation is carried out in small batches. Here, we present a fully automated TMS derivatisation protocol using robotic autosamplers and we also evaluate a commercial software, Maestro available from Gerstel GmbH. Because of automation, there was no waiting time of derivatised samples on the autosamplers, thus reducing degradation of unstable metabolites. Moreover, this method allowed us to overlap samples and improved throughputs. We compared data obtained from both manual and automated TMS methods performed on three different matrices, including standard mix, wine, and plasma samples. The automated TMS method showed better reproducibility and higher peak intensity for most of the identified metabolites than the manual derivatisation method. We also validated the automated method using 114 quality control plasma samples. Additionally, we showed that this online method was highly reproducible for most of the metabolites detected and identified (RSD < 20) and specifically achieved excellent results for sugars, sugar alcohols, and some organic acids. To the very best of our knowledge, this is the first time that the automated TMS method has been applied to analyse a large number of complex plasma samples. Furthermore, we found that this method was highly applicable for routine metabolite profiling (both targeted and untargeted) in any metabolomics laborator

Importance of polyfunctional thiols on semi-industrial Gew\uc3\ubcrztraminer wines and the correlation to technological treatments

Thiol compounds responsible for tropical fruit associated aroma have been extensively studied over the last 20 years. The occurrence of their non-aromatic precursors in grapes and musts is reported largely mainly for the cultivar Sauvignon Blanc. The presence of these thiols as precursors or free molecules in grape, juice, and wine has been reported in several different varieties, suggesting that they are more or less ubiquitous both for Vitis spp. and interspecific hybrids. The biosynthetic pathways resulting in these compounds are yet to be completely elucidated, but, in the meantime, industry needs to improve technological knowledge to better manage winemaking steps to enhance the variety-dependent aroma of wine. In this work, we studied the implications of the use of grape skin tannins\u2014rich and poor in thiol precursors\u2014 on the final content of 3-sulfanylhexan-1-ol (3MH) and its acetate (3MHA) in wine and the effect in terms of sensory appreciability. The evaluation of 36 vinifications carried out in a semi-industrial scale permitted us to prove that only a tannin originally rich in precursors (High), when added to juice at the beginning of fermentation, enhanced both the concentration of precursors in the juice and the final concentration of aromatic thiols in the resultant wine. The 3MH and 3MHA developed as a consequence of the juice supplementation with tannin High and increased pleasantness and typicality of Gew\ufcrztraminer wines. A later supplementation with tannin High at the end of the alcoholic fermentation was sensorially not effective

Conditions promoting effective very high gravity sugarcane juice fermentation

Abstract Background: Applying very high gravity (VHG) fermentation conditions to the sugarcane juice (SCJ) bioethanol industry would improve its environmental and economic sustainability without the need for major infrastructure changes or investments. It could enable a decrease in the consumption of biological and natural resources (cane/ land, water and energy) while maintaining acceptable production parameters. The present study attempts to demonstrate and characterise an efective industrially relevant SCJ-VHG fermentation process. Results: An industry-like SCJ-VHG bioethanol production process with 30 and 35 °Bx broth was employed to investigate the efects of both the yeast strain used and nitrogen source supplementation on process yield, process productivity, biomass viability, glycerol concentration and retention-associated gene expression. Process performance was shown to be variably afected by the diferent process conditions investigated. Highest process efciency, with a 17% (w/v) ethanol yield and only 0.2% (w/v) sugar remaining unfermented, was observed with the Saccharomyces cerevisiae industrial strain CAT-1 in 30 °Bx broth with urea supplementation. In addition, efcient retention of glycerol by the yeast strain was identifed as a requisite for better fermentation and was consistent with a higher expression of glycerol permease STL1 and channel FPS1. Urea was shown to promote the deregulation of STL1 expression, overcoming glucose repression. The consistency between Fps1-mediated ethanol secretion and ethanol in the extracellular media reinforces previous suggestions that ethanol might exit the cell through the Fps1 channel. Conclusions: This work brings solid evidence in favour of the utilisation of VHG conditions in SCJ fermentations, bringing it a step closer to industrial application. SCJ concentrated up to 30 °Bx maintains industrially relevant ethanol production yield and productivity, provided the broth is supplemented with a suitable nitrogen source and an appropriate industrial bioethanol-producing yeast strain is used. In addition, the work contributes to a better understanding of the VHG-SCJ process and the variable efects of process parameters on process efciency and yeast strain response. Keywords: Biofuel, Bioethanol, Sugarcane, Saccharomyces cerevisiae, CAT-1, Very high gravity, Process optimisation, Process sustainabilityB. Monteiro was supported by the Ph.D. Grant 2011/12185-0 from the Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP. P. Ferraz and M. Barroca are supported by the Doctoral Programme in Applied and Environmental Microbiology (DP-AEM) and the FCT by Ph.D. Grants PD/ BD/113814/2015 and PD/BD/113810/2015, respectively. T. Collins thanks the FCT for support through the Investigador FCT Programme (IF/01635/2014). T. Collins and C. Lucas are supported by the strategic programme UID/ BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and the ERDF through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). P. Ferraz, T. Collins and C. Lucas were further funded by the project EcoAgriFood (NORTE-01- 0145-FEDER-000009), supported by the Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio