Cristina Piras

A NMR metabolomics study of the ripening process of the Fiore Sardo cheese produced with autochthonous adjunct cultures

Fiore Sardo (FS) is a traditional Italian raw ewes milk cheese carrying a Protected Designation of Origin (PDO). This study investigated the kinetics of FS cheese ripening by physicochemical parameters, microbial counting, and NMR metabolomics using aqueous extracts. Four Fiore Sardo cheeses, manufactured from milk with deliberately added autochthonous lactic acid bacteria (LAB) or commercial starters were studied during a period of 90days of ripening. Major differences in the metabolic profiles were observed amongst the samples as a function of the adjunct culture utilised. (1)H NMR metabolomics in combination with multivariate data analysis was able to classify cheese samples on the basis of their maturation age and the type of added cultures. These findings lay the metabolic basis for the authentication of Fiore Sardo cheese produced in compliance with PDO specifications which allow the use of only native LAB cultures.

NMR Investigation of Imidazolium-Based Ionic Liquids and Their Aqueous Mixtures

(1)H and (13)C NMR spectroscopy is employed to investigate the interaction of water with two imidazolium-based ionic liquids (ILs), 1-hexyl-3-methylimidazolium bromide ([C(6)mim]Br) and 1-octyl-3-methylimidazolium bromide ([C(8)mim]Br), at IL concentrations well above the critical aggregation concentration (CAC). The results are compared with those of the neat samples. To this aim, a detailed analysis of the changes in the (1)H chemical shifts, (13)C relaxation parameters, and 2D ROESY data due to the presence of water is performed. The results for both neat ILs are consistent with a packed structure where head-to-head, head-to-tail, and tail-to-tail contacts occur and where the site of maximal mobility restriction is at the polar head. At the lowest investigated water content, the presence of water influences mainly the environment around the IL polar head, slowing down the motional dynamics of the aromatic ring with respect to the alkyl chain. At higher water contents this difference diminishes, the motional freedom of the whole molecule increasing. The presence of ROESY cross-peaks between protons in the polar and apolar IL regions, as well as between protons in non-neighboring alkyl groups, at all investigated water contents suggests that the alkyl tails are not fully segregated in hydrophobic domains, as expected for micelle-like structures.

Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract of uncertain origin, which includes ulcerative colitis (UC) and Crohn’s disease (CD). The composition of gut microbiota may change in IBD affected individuals, but whether dysbiosis is the cause or the consequence of inflammatory processes in the intestinal tissue is still unclear. Here, the composition of the microbiota and the metabolites in stool of 183 subjects (82 UC, 50 CD, and 51 healthy controls) were determined. The metabolites content and the microbiological profiles were significantly different between IBD and healthy subjects. In the IBD group, Firmicutes, Proteobacteria, Verrucomicrobia, and Fusobacteria were significantly increased, whereas Bacteroidetes and Cyanobacteria were decreased. At genus level Escherichia, Faecalibacterium, Streptococcus, Sutterella and Veillonella were increased, whereas Bacteroides, Flavobacterium, and Oscillospira decreased. Various metabolites including biogenic amines, amino acids, lipids, were significantly increased in IBD, while others, such as two B group vitamins, were decreased in IBD compared to healthy subjects. This study underlines the potential role of an inter-omics approach in understanding the metabolic pathways involved in IBD. The combined evaluation of metabolites and fecal microbiome can be useful to discriminate between healthy subjects and patients with IBD.

Analysing the effects of frozen storage and processing on the metabolite profile of raw mullet roes using 1H NMR spectroscopy

(1)H NMR spectroscopy was used to investigate changes in the low molecular weight metabolic profile of raw mullet (Mugil spp.) roes during frozen storage and upon processing. NMR data were analysed by Principal Component Analyses (PCA). In the model constructed using frozen roes, no statistical significant metabolic modifications were observed in the first six months of storage, while choline derivatives, dimethylamine, lactate, and most of the free amino acids were identified as changing with statistical significance (p<0.05) in response to frozen storage time of twelve months. The PCA model comparing the metabolic profiles of roes before and after processing showed that the major modifications occurring upon manufacturing were the increase of the choline derivative compounds, uracil, and free amino acids, and a large decrease of taurine, glucose, lactate, and creatine/phosphocreatine. All of the above mentioned modifications reflect the occurrence of chemical/biochemical reactions arising from degradation processes such as lipolysis and proteolysis.

1H NMR metabolite fingerprint and pattern recognition of mullet (Mugil cephalus) bottarga.

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate data analysis (MVA) was used to investigate the molecular components of the aqueous extract of samples of bottarga, that is, salted and dried mullet (Mugil cephalus) roe, manufactured in Sardinia (Italy) from mullets of known and unknown geographical provenience. Principal component analysis (PCA) applied to the processed (1)H NMR spectra indicated that samples tend to cluster according to their geographical origin and also on the basis of storage and manufacturing procedures. The most important metabolites that characterized grouping of samples are the free amino acids methionine (Met), glutamate (Glu), histidine (His), phenylalanine (Phe), tyrosine (Tyr), and isoleucine (Ile); trimethylamine (TMA) and dimethylamine (DMA), both biomarkers of degradation; nucleotides and derivatives; choline (Cho) and phosphorylcholine (P-cho); and lactate (Lac).

Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55-67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7-9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32-39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and β-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log(10)CFU/g) and lactic acid bacteria (3 log(10) CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.

Metabolomic profile in hyperthyroid patients before and after antithyroid drug treatment: Correlation with thyroid hormone and TSH concentration

Hyperthyroidism (HT) is characterized by an intense metabolic impact which affects the lipid, carbohydrate and amino acids metabolism, with increased resting energy expenditure and thermogenesis. Metabolomics is a new comprehensive technique that allows to capture an instant metabolic picture of an organism, reflecting peculiar molecular and pathophysiological states. The aim of the present prospective study was to identify a distinct metabolomic profile in HT patients using 1H NMR spectroscopy before and after antithyroid drug treatment. This prospective study included 15 patients (10 female, 5 male) who were newly diagnosed hyperthyroidism. A nuclear magnetic resonance (1H NMR) based analysis was performed on plasma samples from the same patients at diagnosis (HypT0) and when they achieved euthyroidism (HypT1). The case groups were compared with a control group of 26 healthy volunteers (C). Multivariate statistical analysis was performed with Partial Least Squares-Discriminant Analysis (PLS-DA). PLS-DA identified a distinct metabolic profile between C and untreated hyperthyroid patients (R2X 0.638, R2Y 0.932, Q2 0.783). Interestingly, a significant difference was also found between C and euthyroid patients after treatment (R2X 0.510, R2Y 0.838, Q2 0.607), while similar cluster emerged comparing HypT0vs HypT1 patients. This study shows that metabolomic profile is deeply influenced by hyperthyroidism and this alteration persists after normalization of thyrotropin (TSH) and free thyroid hormone (FT3, FT4) concentration. This suggests that TSH, FT3 and FT4 assays may not be insufficient to detect long lasting peripheral effects of the thyroid hormones action. Further studies are needed to clarify whether and to what extent the evaluation of metabolomics profile may provide relevant information in the clinical management of hyperthyroidism.

Author Correction: Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid, free fatty acids and amino acid levels

BackgroundInflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn’s disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression.ObjectivesThe lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples.MethodsA total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis.ResultsVariations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A2 and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease.ConclusionsGiven good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.