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The human fecal metabolome is increasingly studied to explore the impact of diet and lifestyle on health and the gut microbiome. However, systematic differences and confounding factors related to age, sex, and diet remain largely unknown. In this study, absolute concentrations of fecal metabolites from 205 healthy Danes (105 males and 100 females, 49 ± 31 years old) were quantified using 1H NMR spectroscopy and the newly developed SigMa software. The largest systemic variation was found to be highly related to age. Fecal concentrations of short-chain fatty acids (SCFA) were higher in the 18 years old group, while amino acids (AA) were higher in the elderly. Sex-related metabolic differences were weak but significant and mainly related to changes in SCFA. The concentrations of butyric, valeric, propionic, and isovaleric acids were found to be higher in males compared to females. Sex differences were associated with a stronger, possibly masking, effect from differential intake of macronutrients. Dietary fat intake decreased levels of SCFA and AA of both sexes, while carbohydrate intake showed weak correlations with valeric and isovaleric acids in females. This study highlights some possible demographic confounders linked to diet, disease, lifestyle, and microbiota that have to be taken into account when analyzing fecal metabolome data.

International audience; Effects of fertilization practices, mineral (M) and organo-mineral (OM), on molecular composition of Nero di Troia cultivar grape berries was studied using conventional chemical analysis, Magnetic Resonance Imaging (MRI) and 1H NMR spectroscopy on intact berries and extracts, respectively, and through analysis of yeast species developed on grape skins. Plants vegetative status did not differ between the two fertilization practices, whereas some grape juice chemical characteristics differed in fertilized grapes. MRI provided information on grape berries morphology through weighted images depending on spin-spin (T2) and spin-lattice (T1) relaxation times. T1 values were the highest in OM grape berries. 1H NMR metabolic profile, combined with chemometric analysis, evidenced significant differences for some metabolites (valine, leucine, isoleucine, proline, and malic acid). Furthermore, higher frequency of yeasts genus Starmella sp., isolated from OM grape berries contributed to reinforcing the found results on the physiological response of wine grape Nero di Troia to fertilization.

The human faecal metabolome is complex, but rich in information and allows investigation of the host metabolism as a function of diet and health. The faecal metabolome is still much less explored than the plasma and urine metabolome, and in order to generate comparable data across laboratories and cohorts, standard operating procedures are required. This study evaluates 10 protocols, using different extraction solvents and sample processing methods for measuring the human faecal metabolome using proton nuclear magnetic resonance (1H NMR) spectroscopy. Three solvents: water, methanol, and dimethyl sulfoxide (DMSO) were investigated at varying concentrations for their ability to extract metabolites directly from faecal slurry or after freeze-drying. The protocols were evaluated on four different pools of human feces. The study also demonstrates a novel signature mapping (SigMa) method for rapid and unbiased processing of complex NMR spectra applied for the first time to human faecal metabolomics. The method is provided with a library containing the chemical shift ranges of 81 common faecal metabolites for future unambiguous and rapid faecal metabolite annotations. The result from the 10 faecal extraction protocols were investigated in terms of reproducibility, coverage, and ability to extract low concentration metabolites. The solvent type was shown to induce the highest variation in the data (45.7%) and the water based extractions allowed detection of the greatest number of metabolites and resulted in the highest reproducibility. Direct extraction of faecal slurry was proved to be more reproducible than freeze-drying. In addition, freeze-drying caused a relative loss of short chain fatty acids (SCFA). DMSO was used for the first time to extract faecal metabolites and enabled the detection of certain bile acids. Some derivatives of SCFA were only detected using methanol as solvent.

Proton Nuclear Magnetic Resonance (NMR) spectroscopic analysis of urine generates rich but complex spectra. Retrieving metabolite information from such spectra is challenging due to signal overlapping, chemical shift changes, and large concentration variations of complex urine metabolome. This study demonstrates a new method, Signature Mapping (SigMa), for the rapid and efficient conversion of raw urine NMR spectra into an informative metabolite table. The principle behind SigMa relies on a division of the urine NMR spectra into Signature Signals (SS), Signals of Unknown spin Systems (SUS) and bins of complex unresolved regions (BINS). The method allows simultaneous detection of urinary metabolites in large NMR metabolomics studies using a SigMa chemical shift library and a new automatic peak picking algorithm. For quantification of SS and SUS SigMa uses multivariate curve resolution, while the unresolved inter-SS spectral regions are binned (BINS). SigMa is tested on three human urine 1H-NMR datasets including spiking experiments, and has proven to be extraordinarily efficient, quantitatively reliable and robust.

The introduction of high amounts of cooked poultry offal in mink feed has been associated with health problems in growing mink. Cooking mink feed is a convenient way of reducing microbiological activity, but it may have a negative effect on raw material quality and animal welfare. This study investigates growth and health of mink fed raw or cooked poultry offal and describes urinary and blood plasma metabolic changes related to the feeding. A total of 65 male mink were divided in three feeding groups, two fed cooked offal and one group fed raw offal, and the plasma and urine samples were collected at 3 time points during the growth. Both bio-fluids and feed samples were measured by 1H NMR spectroscopy and resulted metabolomics data were analysed using univariate and multivariate statistical methods that revealed dominating effect of the mink growth stages and to a less extent the feeding regime. Metabolome differences in relation to low body mass index (BMI) and kidney lesions were observed in plasma. Disease and decrease in BMI was associated with high creatinine and dimethylglycine content in plasma. These molecules were also particularly indicative of the cooked feeds. Moreover, low urinary taurine levels were also associated with disease and low BMI. Individual mink appeared to show negative effects of the cooked feed diet, including impaired growth and gross pathological lesions involving the kidneys. This may be related to the absorption of essential metabolites such as amino acids and fats, necessary for mink growth, that are negatively impacted by the cooking process.

The human fecal metabolome is increasingly studied to explore the impact of diet and lifestyle on health and the gut microbiome. However, systematic differences and confounding factors related to age, sex, and diet remain largely unknown. In this study, absolute concentrations of fecal metabolites from 205 healthy Danes (105 males and 100 females, 49 ± 31 years old) were quantified using

The human faecal metabolome is complex, but rich in information and allows investigation of the host metabolism as a function of diet and health. The faecal metabolome is still much less explored than the plasma and urine metabolome, and in order to generate comparable data across laboratories and cohorts, standard operating procedures are required. This study evaluates 10 protocols, using different extraction solvents and sample processing methods for measuring the human faecal metabolome using proton nuclear magnetic resonance (

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state‐of‐the‐art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

Proton Nuclear Magnetic Resonance (NMR) spectroscopic analysis of urine generates rich but complex spectra. Retrieving metabolite information from such spectra is challenging due to signal overlapping, chemical shift changes, and large concentration variations of complex urine metabolome. This study demonstrates a new method, Signature Mapping (SigMa), for the rapid and efficient conversion of raw urine NMR spectra into an informative metabolite table. The principle behind SigMa relies on a division of the urine NMR spectra into Signature Signals (SS), Signals of Unknown spin Systems (SUS) and bins of complex unresolved regions (BINS). The method allows simultaneous detection of urinary metabolites in large NMR metabolomics studies using a SigMa chemical shift library and a new automatic peak picking algorithm. For quantification of SS and SUS SigMa uses multivariate curve resolution, while the unresolved inter-SS spectral regions are binned (BINS). SigMa is tested on three human urine

The aim of this study was to investigate the alteration of the human urine metabolome by means of diet and to compare the metabolic effects of the nutritionally healthy New Nordic Diet (NND) with an Average Danish Diet (ADD). The NND was designed a decade ago by scientists and chefs, based on local and sustainable foods, including fish, shellfish, vegetables, roots, fruit, and berries. The NND has been proven to lower blood pressure, reduce glycemia, and lead to weight loss.The human urine metabolome was measured by untargeted proton nuclear magnetic resonance spectroscopy in samples from 142 centrally obese Danes (20-66 years old), randomized to consume the ADD or the NND. The resulting metabolomics data was processed and analyzed using advanced multivariate data analysis methods to reveal effects related to the design factors, including diet, season, sex, and changes in body weight.Exploration of the nuclear magnetic resonance profiles revealed unique metabolite markers reflecting changes in protein and carbohydrate metabolism between the two diets. Glycine betaine, glucose, trimethylamine N-oxide and creatinine were increased in urine of the individuals following the NND compared with the ADD population, whereas relative concentrations of tartrate, dimethyl sulfone, and propylene glycol were decreased. Propylene glycol had a strong association with the homeostatic model assessment for insulin resistance in the NND group. The food intake biomarkers found in this study confirm the importance of these as tools for nutritional research.Findings from this study provided new insights into the effects of a healthy diet on glycemia, reduction of inflammation, and weight loss among obese individuals, and alteration of the gut microbiota metabolism.

Objectives The aim of this study was to investigate the alteration of the human urine metabolome by means of diet and to compare the metabolic effects of the nutritionally healthy New Nordic Diet (NND) with an Average Danish Diet (ADD). The NND was designed a decade ago by scientists and chefs, based on local and sustainable foods, including fish, shellfish, vegetables, roots, fruit, and berries. The NND has been proven to lower blood pressure, reduce glycemia, and lead to weight loss. Methods The human urine metabolome was measured by untargeted proton nuclear magnetic resonance spectroscopy in samples from 142 centrally obese Danes (20–66 years old), randomized to consume the ADD or the NND. The resulting metabolomics data was processed and analyzed using advanced multivariate data analysis methods to reveal effects related to the design factors, including diet, season, sex, and changes in body weight. Results Exploration of the nuclear magnetic resonance profiles revealed unique metabolite markers reflecting changes in protein and carbohydrate metabolism between the two diets. Glycine betaine, glucose, trimethylamine N-oxide and creatinine were increased in urine of the individuals following the NND compared with the ADD population, whereas relative concentrations of tartrate, dimethyl sulfone, and propylene glycol were decreased. Propylene glycol had a strong association with the homeostatic model assessment for insulin resistance in the NND group. The food intake biomarkers found in this study confirm the importance of these as tools for nutritional research. Conclusions Findings from this study provided new insights into the effects of a healthy diet on glycemia, reduction of inflammation, and weight loss among obese individuals, and alteration of the gut microbiota metabolism.

Scope: According to Eurostat 2016, approximately 119 million European citizens live at-risk-of-poverty (ROP). This subpopulation is highly diverse by ethnicity, age, and culture in the different EU states, but they all have in common a low income that could represent an increased risk of nutrient deficiencies due to poor nutritional habits. This study aims to investigate the human urine metabolome in the search of common biomarkers representing dietary deficiencies amongst European populations at ROP. Methods and results: 2732 urine samples were collected from 1391 subjects across five different European countries, including the United Kingdom, Finland, Italy, Lithuania, and Serbia, and analyzed using 1H-NMR spectroscopy. The resulting urine metabolome data were explored according to study design factors including economic status, country, and gender. Conclusion: Partitioning of the effects derived from the study design factors using ANOVA-simultaneous component analysis (ASCA) revealed that country and gender effects were responsible for most of the systematic variation. The effect of economic status was, as expected, much weaker than country and gender, but more pronounced in Lithuania than in other countries. Citrate and hippurate were among the most powerful ROP biomarkers. The possible relationship between these markers and nutritional deficiencies amongst the ROP population is discussed.

5th International Conference on Foodomics - From Data to Knowledge -- JAN 10-12, 2018 -- Cesena, ITALY, WOS: 000456758800042, PubMed ID: 30599953, In this work, the effects of maturation time and simulated gastrointestinal digestion on the molecular and peptide profiles of "Bresaola Valtellina" were assessed through the foodomics approach, in this case food proteomics and peptidomics combined to other analytical and biological assays, aiming at depicting a holistic food quality. Human digestion of this Italian cured meat product was simulated using an in vitro static protocol and the degree of proteolysis and the in vitro bioactivity of the soluble free compounds in the digestates were evaluated by biochemical assays, e.g. SDS-PAGE, size exclusion HPLC, HPLC/MS, 1H NMR, enzymatic and antioxidant activities. The obtained results demonstrated that in vitro gastrointestinal digestion contributed to a considerable release of myofibrillar proteins by the muscle tissue. Data from SDS-PAGE, peptidomic and size exclusion HPLC assays showed that the in vitro digestion largely degraded proteins of muscle tissue to peptides smaller than 250 Da. The released peptides were likely responsible for the inhibitory activity on amylolytic enzymes and for the antioxidant properties elicited by the gastric digestates of Bresaola. Overall, the results demonstrated the negligible role of ripening in making meat proteins more bioaccessible, whereas they confirmed the highly in vitro digestibility of meat proteins from Bresaola. This study represents a new approach merging proteomics and foodomics to evaluate the effect of ripening and in vitro digestion on the bioactivity and bioaccessibility of proteins and peptides of meat products., COST actionEuropean Cooperation in Science and Technology (COST) [FA1005 INFOGEST], The authors participate in the COST action FA1005 INFOGEST (http://www.cost-INFOGEST.eu/).

Quality definition of food includes several complex factors like physical, compositional and microbial features, modifications induced by technological processes or storage, nutritional value and safety. Foodomics is a holistic approach applying omics technologies to observe food along the entire production/consumption chain. In the present review, we present key applications of nuclear magnetic resonance in foodomics described in the 2012–2015 period, in the quest for robust and thorough information required by the scientific community. In doing so, we summarize the issues connected to food traceability and authenticity, composition and physical characteristics, processing and storage and health, that mostly impact food quality.

The identification and validation of food intake biomarkers (FIBs) in human biofluids is a key objective for the evaluation of dietary intake. We report here the analysis of the GC-MS and 1H-NMR metabolomes of serum samples from a randomized cross-over study in 11 healthy volunteers having consumed isocaloric amounts of milk, cheese, and a soy drink as non-dairy alternative. Serum was collected at baseline, postprandially up to 6 h, and 24 h after consumption. A multivariate analysis of the untargeted serum metabolomes, combined with a targeted analysis of candidate FIBs previously reported in urine samples from the same study, identified galactitol, galactonate, and galactono-1,5-lactone (milk), 3-phenyllactic acid (cheese), and pinitol (soy drink) as candidate FIBs for these products. Serum metabolites not previously identified in the urine samples, e.g., 3-hydroxyisobutyrate after cheese intake, were detected. Finally, an analysis of the postprandial behavior of candidate FIBs, in particular the dairy fatty acids pentadecanoic acid and heptadecanoic acid, revealed specific kinetic patterns of relevance to their detection in future validation studies. Taken together, promising candidate FIBs for dairy intake appear to be lactose and metabolites thereof, for lactose-containing products, and microbial metabolites derived from amino acids, for fermented dairy products such as cheese.

Introduction Mixed-linkage (1→3),(1→4)-β-d-glucans (BG) reduce cholesterol level and insulin response in humans. Despite this, their role in human metabolism and a mode of action remains largely unknown. Objectives To investigate the effects of three structurally different BG on human fecal metabolome in a full cross-over intervention using GC-MS metabolomics. Methods Over three weeks of intervention, young healthy adults received food supplemented with BG from oat, two different BG from barley or a non-fiber control in a full cross-over design. Untargeted metabolomics and short chain fatty acid analysis was performed on day three fecal samples. ANOVA-simultaneous component analysis was applied to partition the data variation according to the study design, and PLS-DA was used to select most discriminative metabolite markers. Results Univariate and multivariate data analysis revealed a dominating effect of inter-individual variances followed by a gender effect. Weak effects of BG intake were identified including an increased level of gamma-amino-butyrate and palmitoleic acid in males and a decreased level of enterolactone in females. Barley and oat derived BG were found to influence the human fecal metabolome differently. Barley BG increased the relative level of formate in males and isobutyrate, isovalerate, 2-methylbutyrate in females. In total 15, 3 and 11 human fecal metabolites were significantly different between control vs. BG, control vs. oat BG, and barley BG vs. oat BG, respectively. Conclusions The study show that human fecal metabolome largely reflects individual (∼28% variation) and gender (∼15% variation) differences, whereas the treatment effect of the BG (∼8% variation) only manifests in a few key metabolites (primarily by the metabolites: d-2-aminobutyric acid, palmitoleic acid, linoleic acid and 11-eicosenoic acid). Electronic supplementary material The online version of this article (doi:10.1007/s11306-017-1247-2) contains supplementary material, which is available to authorized users.

The measurement of food intake biomarkers (FIBs) in biofluids represents an objective tool for dietary assessment. FIBs of milk and cheese still need more investigation due to the absence of candidate markers. Thus, an acute intervention study has been performed to sensitively and specifically identify candidate FIBs. Eleven healthy male and female volunteers participated in the randomised, controlled crossover study that tested a single intake of milk and cheese as test products, and soy based drink as a control. Urine samples were collected at baseline and up to 24h at distinct time intervals (0-1, 1-2, 2-4, 4-6, 6-12 and 12-24h) and were analysed using an untargeted multi-platform approach (GC-MS and 1H-NMR). Lactose, galactose and galactonate were identified exclusively after milk intake while for other metabolites (allantoin, hippurate, galactitol and galactono-1,5-lactone) a significant increase has been observed. Urinary 3-phenyllactic acid was the only compound specifically reflecting cheese intake although alanine, proline and pyroglutamic acid were found at significantly higher levels after cheese consumption. In addition, several novel candidate markers for soy drink were identified such as pinitol and trigonelline. Together, these candidate FIBs of dairy intake could serve as a basis for future validation studies under free-living conditions.

During the recent decades, proteomics and metabolomics approaches have been extensively integrated in methods for the assessment of meat quality. The high-throughput nature of these omics sciences has in fact been exploited in many different applications, ranging from the genetic fingerprinting of specific breeds and traits to the definition of phenotypes obtained with specific breeding programs or feeding and rearing conditions, to the study of the transformations occurring during and after meat processing. The present chapter aims at describing the main proteomics and metabolomics platforms and methods employed for the evaluation of meat quality. Although the literature mainly reports research studies carried out by applying such omics methods to pork and beef, this chapter plots future scenarios for the inclusion of proteomics and metabolomics in protocols specifically designed for investigations on poultry meat.

Introduction: The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch. Objectives: This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome. Methods: The effects of the genetic modification upon the grape metabolome were evaluated through 1H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one. Results: The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration. Conclusion: NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.

The increasing demand for natural foods and beverages, i.e. prepared by excluding synthetic chemicals along the whole production chain, has boosted the adoption of organic and biodynamic cultivation methods which are based on protocols avoiding use of synthetic pesticides. This trend is striking in viticulture, since wine production is largely shaped by the varying drinking attitudes of environment-friendly consumers. Using 1 H NMR, the compositions of grape berries, collected at harvest in 2009 and 2011, in experimental plots cultivated either with biodynamic or organic methods, were compared. Although the analysis provides a comprehensive metabolic profile of berries, the resulting distinctive pattern consists of a few molecules. Lower content of sugars, coumaric and caffeic acids, as well as higher amount of γ-aminobutyric acid (GABA) were observed in biodynamic grapes. The 1 H NMR foodomics approach evidenced a diverse fruit metabolome that could be associated to a different physiological response of plants to the agronomic environment.

Food science has been increasingly linked to other fields such as medicine, veterinary science, agriculture, biology, and genetics. Metabolomics can assist those different fields by identifying connections between food quality and health, while accounting for food quality changes that result from human and environmental perturbations on the food metabolome. This chapter summarises some methods for applying metabolomics in the evaluation of food quality as it relates to genetic selection and modifications, different growing (e.g. organic vs. conventional) and rearing conditions, geographic origin, food manufacturing protocols, and the effect of digestion on the nutrient availability.

The analysis of the metabolome has become particularly important in human studies, allowing the identification of typical and atypical metabolites in determined biofluids and tissues. This is clearly fundamental for medical investigations, since it could be possible to assess the presence or absence of certain diseases and syndromes or risk conditions. Another widespread use of the metabolomic approach is the research on specific dietary effects on the metabolic profile. It is necessary to take research further and investigate the presence of determined food molecules in fluids or tissues from individuals without a controlled dietary intervention. NMR spectroscopy techniques can be very useful in different nutrimetabonomics approaches, thanks to the quickness, high-throughput efficacy and high-reproducibility of its methods. In this paper the two main nutrimetabolomic strategies were highlighted, both greatly aided by the use of NMR spectroscopy. The first one requires a direct intervention on a sample population in order to assess the direct effects on the metabolome after the intake of a particular food product or category. In the other technique, instead, observational studies in free-eating populations are carried out. At first nutritional data is analysed in order to find possible dietary patterns, then these have to be proved by the inspection of biofluid spectra, in order to find molecules capable in discriminating among the dietary behaviours.

Nuclear Magnetic Resonance (NMR) methodologies offer a comprehensive characterization of foodstuff owing to the possibility to study a sample from different points of view including structural, compositional, functional, morphological etc. aspects. High resolution NMR spectroscopy applied to semi-solid food samples or to extracts in solution is used to determine the foodstuff composition. Here, some features of high resolution NMR methodologies related to food analysis such as quantitative analysis, chemometrics, and use of databases are included. Other NMR methodologies such as relaxometry and imaging described in this chapter give precious information regarding the morphology and texture of intact food samples.