Metabolic fingerprinting of <i>Lactobacillus paracasei</i>:the optimal quenching strategy

BACKGROUND: Quenching in cold buffered methanol at −40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline. RESULTS: Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in Lactobacillus paracasei subsp. paracasei (L. paracasei). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the L. paracasei cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the L. paracasei culture, fast filtration took several minutes, which is far from ideal for metabolites with high intracellular turnover rates. CONCLUSION: The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

Introduction Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. Materials and Methods The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow. Conclusions Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.The authors are thankful to Mia Jullig for assistance with Fig. 2. Callaghan Innovation and Bioresource Processing Alliance provided PhD stipends for James Daniell and Ninna Granucci respectively.info:eu-repo/semantics/publishedVersio

Use of metabolomics for the identification and validation of clinical biomarkers for preterm birth:Preterm SAMBA

Made available in DSpace on 2018-12-11T17:29:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-08-08Background: Spontaneous preterm birth is a complex syndrome with multiple pathways interactions determining its occurrence, including genetic, immunological, physiologic, biochemical and environmental factors. Despite great worldwide efforts in preterm birth prevention, there are no recent effective therapeutic strategies able to decrease spontaneous preterm birth rates or their consequent neonatal morbidity/mortality. The Preterm SAMBA study will associate metabolomics technologies to identify clinical and metabolite predictors for preterm birth. These innovative and unbiased techniques might be a strategic key to advance spontaneous preterm birth prediction. Methods/design: Preterm SAMBA study consists of a discovery phase to identify biophysical and untargeted metabolomics from blood and hair samples associated with preterm birth, plus a validation phase to evaluate the performance of the predictive modelling. The first phase, a case-control study, will randomly select 100 women who had a spontaneous preterm birth (before 37 weeks) and 100 women who had term birth in the Cork Ireland and Auckland New Zealand cohorts within the SCOPE study, an international consortium aimed to identify potential metabolomic predictors using biophysical data and blood samples collected at 20 weeks of gestation. The validation phase will recruit 1150 Brazilian pregnant women from five participant centres and will collect blood and hair samples at 20 weeks of gestation to evaluate the performance of the algorithm model (sensitivity, specificity, predictive values and likelihood ratios) in predicting spontaneous preterm birth (before 34 weeks, with a secondary analysis of delivery before 37 weeks). Discussion: The Preterm SAMBA study intends to step forward on preterm birth prediction using metabolomics techniques, and accurate protocols for sample collection among multi-ethnic populations. The use of metabolomics in medical science research is innovative and promises to provide solutions for disorders with multiple complex underlying determinants such as spontaneous preterm birth.University of Campinas (UNICAMP) School of Medical Sciences Department of Obstetrics and Gynecology, R. Alexander Fleming, 101University of Auckland Gravida: National Centre for Growth and Development Liggins InstituteUniversity College Cork Irish Centre for Fetal and Neonatal Translational Research (INFANT) Department of Obstetrics and GynaecologyUniversity of Auckland South Auckland Clinical School Faculty of Medical and Health SciencesUniversity of Auckland School of Biological SciencesUniversity of Campinas (UNICAMP) LNBio-Brazilian Biosciences National Laboratory and School of Medical SciencesSchool of Medical Sciences University of CampinasLNBioSchool of Medicine of Botucatu UNESPSchool of Medicine Federal University of Rio Grande do SulSchool of Medicine Federal University of PernambucoSchool of Medicine Federal University of CearáKing's College London and King's Health PartnersMaternal and Fetal Health Research Centre University of ManchesterUniversity of LeedsUniversity of AdelaideSchool of Medicine of Botucatu UNES

Rapid Quantification of Major Volatile Metabolites in Fermented Food and Beverages Using Gas Chromatography-Mass Spectrometry

Here we present a method for the accurate quantification of major volatile metabolites found in different food and beverages, including ethanol, acetic acid and other aroma compounds, using gas chromatography coupled to mass spectrometry (GC-MS). The method is combined with a simple sample preparation procedure using sodium chloride and anhydrous ethyl acetate. The GC-MS analysis was accomplished within 4.75 min, and over 80 features were detected, of which 40 were positively identified using an in-house and a commercialmass spectrometry (MS) library. We determined different analytical parameters of these metabolites including the limit of detection (LOD), limit of quantitation (LOQ) and range of quantification. In order to validate the method, we also determined detailed analytical characteristics of five major fermentation end products including ethanol, acetic acid, isoamyl alcohol, ethyl-L-lactate and, acetoin. The method showed very low technical variability for the measurements of these metabolites in different matrices (&lt;3%) with an excellent accuracy (100% ± 5%), recovery (100% ± 10%), reproducibility and repeatability [Coefficient of variation (CV) 1–10%)]. To demonstrate the applicability of the method, we analysed different fermented products including balsamic vinegars, sourdough, distilled (whisky) and non-distilled beverages (wine and beer)

Extracellular Microbial Metabolomics: The State of the Art

Microorganisms produce and secrete many primary and secondary metabolites to the surrounding environment during their growth. Therefore, extracellular metabolites provide important information about the changes in microbial metabolism due to different environmental cues. The determination of these metabolites is also comparatively easier than the extraction and analysis of intracellular metabolites as there is no need for cell rupture. Many analytical methods are already available and have been used for the analysis of extracellular metabolites from microorganisms over the last two decades. Here, we review the applications and benefits of extracellular metabolite analysis. We also discuss different sample preparation protocols available in the literature for both types (e.g., metabolites in solution and in gas) of extracellular microbial metabolites. Lastly, we evaluate the authenticity of using extracellular metabolomics data in the metabolic modelling of different industrially important microorganisms

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells

Metabolic response of Candida albicans to phenylethyl alcohol under hyphae-inducing conditions.

Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD(+)/NADH and NADP(-/)NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis