Application of Non-negative Matrix Factorization to LC/MS data

International audienceLiquid Chromatography-Mass Spectrometry (LC/MS) provides large datasets from which one needs to extract the relevant information. Since these data are made of non-negative mixtures of non-negative mass spectra, non-negative matrix factorization (NMF) is well suited for its processing, but it has barely been used in LC/MS. Also, these data are very difficult to deal with since they are usually contaminated with non-Gaussian noise and the intensities vary on several orders of magnitude. In this article, we show the feasibility of the NMF approach on these data. We also propose an adaptation of one of the algorithms aiming at specifically dealing with LC/MS data. We finally perform experiments and compare standard NMF algorithms on both simulated data and an annotated LC/MS dataset. This lets us evaluate the influence of the noise model and the data model on the recovery of the sources

The rennet coagulation mechanisms of a concentrated casein suspension as observed by PFG-NMR diffusion measurements

International audiencePulsed field gradient - nuclear magnetic resonance (PFG-NMR) was used to monitor the diffusion of caseins throughout the rennet coagulation of a highly concentrated casein suspension. Two types of casein species were distinguished according to their diffusing properties and attributed to the dissolved casein fraction, i.e. dissociated caseins and caseinomacropeptides (CMP), and casein particles, respectively. The NMR signal intensity coming from the dissolved casein fraction, which is related to the number of protons it contains, increased all along the experiment whereas the opposite tendency was observed for casein particles. This was explained by the increasing amount of CMP in the whey and the reciprocal loss of protons contained by casein particles, and used to quantitatively estimate the kinetics of the chymosin action. The diffusion of the dissolved casein fraction remained nearly constant during the coagulation process whereas, as revealed by rheometry measurements, the diffusion of casein particles was very sensitive to the structural reorganization of the sample. It decreased by about 35 % during the sol-gel transition and increased in similar proportions during the succeeding rise in gel porosity. Our results also provide different types of information on the respective behaviors of dissociated caseins and casein particles during the course of the process, the most remarkable one being that all the casein particles did not aggregate during the sol-gel transition of our sample. This strongly suggests that the rennet gelation of a concentrated dairy solution may be better visualized by the formation of a network backbone during the sol-gel transition which is thereafter reinforced upon further incorporation of casein particles

The rennet coagulation mechanisms of a concentrated casein suspension as observed by PFG-NMR diffusion measurements

International audiencePulsed field gradient - nuclear magnetic resonance (PFG-NMR) was used to monitor the diffusion of caseins throughout the rennet coagulation of a highly concentrated casein suspension. Two types of casein species were distinguished according to their diffusing properties and attributed to the dissolved casein fraction, i.e. dissociated caseins and caseinomacropeptides (CMP), and casein particles, respectively. The NMR signal intensity coming from the dissolved casein fraction, which is related to the number of protons it contains, increased all along the experiment whereas the opposite tendency was observed for casein particles. This was explained by the increasing amount of CMP in the whey and the reciprocal loss of protons contained by casein particles, and used to quantitatively estimate the kinetics of the chymosin action. The diffusion of the dissolved casein fraction remained nearly constant during the coagulation process whereas, as revealed by rheometry measurements, the diffusion of casein particles was very sensitive to the structural reorganization of the sample. It decreased by about 35 % during the sol-gel transition and increased in similar proportions during the succeeding rise in gel porosity. Our results also provide different types of information on the respective behaviors of dissociated caseins and casein particles during the course of the process, the most remarkable one being that all the casein particles did not aggregate during the sol-gel transition of our sample. This strongly suggests that the rennet gelation of a concentrated dairy solution may be better visualized by the formation of a network backbone during the sol-gel transition which is thereafter reinforced upon further incorporation of casein particles

Semi-automated solid-phase extraction method for studying the biodegradation of ochratoxin A by human intestinal microbiota

A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9 mL of air), the cartridge was rinsed with 5-mL H2O/ACN (80:20, v/v), before eluting the compounds with 3 x 1 mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled: this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72 h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin alpha, open ochratoxin A and ochratoxin B. (C) 2012 Elsevier B.V. All rights reserved

Probing substrate-product relationships by natural abundance deuterium 2D NMR spectroscopy in liquid-crystalline solvents: epoxidation of linoleate to vernoleate by two different plant enzymes

International audienceNatural abundance deuterium 2D NMR spectroscopy in weakly ordering, polypeptide chiral liquid crystals is a powerful technique that enables determination of enantiotopic isotopic ratios (H-2/H-1) (i) at the methylene groups of long-chain fatty acids. This technique has been used to study the bioconversion of linoleic acid to vernoleic acid with the objective of establishing the in-vivo site-specific fractionation of H-2 associated with this process. The fractionation pattern was investigated in Euphorbia lagascae and Vernonia galamensis, plants that use different enzyme systems to perform the Delta(12)-epoxidation: a cytochrome P450 monooxygenase in the former and a di-iron dioxygenase in the latter. The specific interest in this study was to ascertain whether different (H-2/H-1) (i) isotopic ratios in substrate and product might reflect distinct features of the nature of the reaction centre. However, both the linoleate (substrate) samples and both vernoleate (product) samples isolated from the seed oils of the two plants had remarkably similar H-2 isotope profiles, with selection against H-2 in the positions around the Delta(12)-epoxidation site. This is interpreted as indicating that, despite differences in the form in which the activated Fe is presented and in the architecture of the active site, the (H-2/H-1) (i) isotopic pattern is determined by features common to the reaction. It is suggested that the effects acting as the overall determinants of the final (H-2/H-1) (i) distribution in the product are the encumbrance of the active site pocket and constraints to conformational readjustment during the linoleate to vernoleate transformation

Metabolic fate of ochratoxin A as a coffee contaminant in a dynamic simulator of the human colon

Ochratoxin A (OTA) is a mycotoxin frequently encountered in coffee. The relevance of this contaminant in the colon upon digestion necessitates a study on its interaction with colon microbiota. Here, the fate of OTA during colon digestion was investigated using a dynamic simulator of the human gut. The influence of coffee as a food matrix was taken into account, as it may affect the colonic microbial ecosystem and, consequently, the fate of OTA. Biodegradation was followed by measuring OTA concentration over time, and by screening for several possible metabolites, using LC-ESI-MS and HRMS. The descending colon was found to be the main site of OTA biodegradation. Two metabolites, ochratoxin a and ochratoxin B, were identified, suggesting that biodegradation by gut microbiota is beneficial for the host, as they are considered less toxic than OTA. The extent of biodegradation was reduced in the presence of the coffee matrix, possibly due to competition for available carbon sources. Effects of OTA and the coffee matrix on the microbial ecosystem were contrasting. While OTA caused a specific, but lasting loss, of the beneficial species Lactobacillus reuteri, coffee temporarily altered the fermentation pattern towards lower ammonia and higher acetate and propionate production, likely due to its dietary fibre content. (C) 2013 Elsevier Ltd. All rights reserved