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Avocado residues, such as seeds, peels, and leaves, are waste generated by the agri-food industry in large quantities since avocado is a fruit of high 4nutritional value consumed in much of the world. However, the increase in its waste is an environmental problem, so its use represents a viable alternative to this problem. It has been shown that these residues are a great source of bioactive compounds such as phenols, tannins, anthocyanins, and flavonoids, among others, whose application in areas such as health is of great importance since these compounds have characteristics due to their antioxidant nature that give them biological properties such as anticancer, antiviral, antimicrobial, antioxidant, hypoglycemic, and others that have been studied to prevent or counteract diseases and that will be described in this chapter.

Metabolomics is a valuable tool to address different questions in food science and nutrition. It covers different strategies depending on the goal of the study. Among the different strategies in metabolomics, metabolic profiling focuses on a subset of biologically relevant metabolites, structurally related or related to a specific metabolic pathway. The choice and the practice of sample preparation will have a major impact on the outcome of the study, as this will directly affect the observed metabolite content. Moreover, it is required to ensure that the metabolites of interest are present in a form compatible with the analytical system. Among the analytical platforms currently used in metabolomics, mass spectrometry has become a choice method for metabolite analysis due to its capacity of performing sensitive and reliable quantifications of a large number of metabolites. In this chapter, we will focus on the current strategies to study the polar fraction of the metabolome in biological samples.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

Nowadays, there is great interest in the discovery of food compounds that might inhibit gut microbial TMA production from its methylamine precursors. In this work, an innovative novel screening strategy capable of rapidly determining the differences in the metabolic response of Klebsiella pneumoniae, a bacteria producing TMA under aerobic conditions, to a library of extracts obtained from food and natural sources was developed. The proposed high-throughput screening (HTS) method combines resazurin reduction assay in 384-well plates and Gaussian Processes as a machine learning tool for data processing, allowing for a fast, cheap and highly standardized evaluation of any interfering effect of a given compound or extract on the microbial metabolism sustained by L-carnitine utilization. As a proof-of-concept of this strategy, a pilot screening of 39 extracts and 6 pure compounds was performed to search for potential candidates that could inhibit in vitro TMA formation from L-carnitine. Among all the extracts tested, three of them were selected as candidates to interfere with TMA formation. Subsequent in vitro assays confirmed the potential of oregano and red thyme hexane extracts (at 1 mg mL−1) to inhibit TMA formation in bacterial lysates. In such in vitro assay, the red thyme extract exerted comparable effects on TMA reduction (∼40%) as 7.5 mM meldonium (∼50% TMA decrease), a reported L-carnitine analogue. Our results show that metabolic activity could be used as a proxy of the capacity to produce TMA under controlled culture conditions using L-carnitine to sustain metabolism.

There is a growing body of clinical evidence that supports a strong association between elevated circulating trimethylamine N-oxide (TMAO) levels with increased risk of developing adverse cardiovascular outcomes such as atherosclerosis and thrombosis. TMAO is synthesized through a meta-organismal stepwise process that involves (i) the microbial production of TMA in the gut from dietary precursors and (ii) its subsequent oxidation to TMAO by flavin-containing monooxygenases in the liver. Choline, L-carnitine, betaine, and other TMA-containing compounds are the major dietary precursors of TMA. TMAO can also be absorbed directly from the gastrointestinal tract after the intake of TMAO-rich foods such as fish and shellfish. Thus, diet is an important factor as it provides the nutritional precursors to eventually produce TMAO. A number of studies have attempted to associate circulating TMAO levels with the consumption of diets rich in these foods. On the other hand, there is growing interest for the development of novel food ingredients that reduce either the TMAO-induced damage or the endogenous TMAO levels through the interference with microbiota and host metabolic processes involved in TMAO pathway. Such novel functional food ingredients would offer great opportunities to control circulating TMAO levels or its effects, and potentially contribute to decrease cardiovascular risk. In this review we summarize and discuss current data regarding the effects of TMA precursors-enriched foods or diets on circulating TMAO levels, and recent findings regarding the circulating TMAO-lowering effects of specific foods, food constituents and phytochemicals found in herbs, individually or in extracts, and their potential beneficial effect for cardiovascular health.

Grouping individuals according to their metabolic capacities (metabotyping) has caused a shift from individualised to grouped treatments for the optimisation of nutritional interventions. Several studies have reported a stratification of patients into metabolic clusters after the intake of certain foods, of which polyphenols seem to be mostly associated with metabotypes. Despite this, there is a lack of metabotyping studies regarding wine consumption. In this context, the human urinary metabolome of healthy volunteers (n=41) was explored by means of a non-targeted metabolomic approach after an intervention with red wine (250 mL/day, 28 days). Three clusters of volunteers based on their relative production of phenolic metabolites were perceived , and the compounds responsible for this clustering were identified. To our knowledge, this is the first time that different urinary metabotypes have been described in healthy volunteers after moderate red wine consumption. Our findings suggest that stratification of individuals in clinical trials according to their metabotype is necessary to fully understand the health effects of wine polyphenols.

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.

Metabolomics is a systems biology tool useful in the study of the plurality of chemical processes involving intermediates and products of metabolism within an organism, cell or tissue. Food metabolomics can be defined as the application of metabolomics in food systems “from farm to human”. The application of metabolomics technologies has had a significant impact on food research and has further improved the sensitivity demanded by existing food quality/safety legislation. Metabolomics has also opened up new opportunities for biomarker food intake discovery and new metabolite biomarkers identified in body fluids following the consumption of various foods, ingredients, meals, or diets. In nutrition research, metabolomics also aims to understand the correlation between diet or food ingredients and their preventive or health-promoting effects. These exciting possibilities would not be possible in the absence of the huge advances in analytical technologies, including nuclear magnetic resonance and mass spectrometry-based techniques. When coupled with chemometric methods and spectral databases for the statistical analysis and biological interpretation of complex metabolomic data, food metabolomics becomes a powerful investigative tool.

The potential of capillary electrophoresis-mass spectrometry (CE-MS) for metabolomics is demonstrated through the analysis of metabolites from human HT29 colon cancer cells treated and non-treated with dietary polyphenols. Prior to CE-MS analysis, four different metabolite purification strategies are investigated. Namely, the results obtained after methanol deproteinization, ultrafiltration, and two solid-phase extraction methods using C18 and polymer-based cartridges are described. These generic methods can have broad applications to analyze metabolites in a large variety of matrices and fields, including the new Foodomics area.

The transcriptome can be defined as the complete set of RNA transcripts produced by the genome at any one time, and transcriptomics as the study of these transcripts. Recent advances in high-throughput technologies have made available groundbreaking methods that enable the characterization of transcriptomes in great detail. The most relevant application of transcriptomic technologies in modern food science include the transcriptome characterization and gene expression analysis in individual organisms, such as food crops, food animals, foodborne pathogens, fermenting microorganisms, and probiotics. As next-generation genome sequencing technologies mature, RNA-seq, a method to detect the presence and quantity of RNA in a given sample, combined with bioinformatics is gaining traction in the investigation of topics in food science that were inconceivable only few years ago (e.g. identification of novel genes not possible previously due to limitations of earlier technologies). Also, this next-generation technology is expected to revolutionize the food microbiology research field by allowing the time- and cost-effective analysis of food microbial communities.

Elevated level of trimethylamine N-oxide (TMAO) in plasma has been recently associated with the development or propagation of atherosclerosis and other adverse cardiovascular events. TMAO is generated via a metaorganismal pathway that starts with conversion of dietary precursors (choline, L-carnitine, etc.) into trimethylamine (TMA) by gut microbiota, followed by host hepatic enzymes of the flavin-containing monooxygenase family. The discovery of novel dietary compounds able to interfere with the microbial TMA formation would be an innovative approach for reduction of risk for cardiovascular diseases. To date there have been no reports of dietary compounds or natural extracts able to inhibit the activity of carnitine monooxygenase, a microbial enzyme that features the conversion of L-carnitine to TMA. In this work we present a methodology that combines resazurin-based microplate assay with a fast and cost-effective CE-UV method for the high-throughput assessment of carnitine monooxygenase inhibitory potential of a collection of natural extracts. For this purpose, a strain of Klebsiella pneumoniae, isolated from human gut, and capable of producing TMA from Lcarnitine in vitro, was used. Here, we demonstrate the versatile applicability of CE-UV by tracking TMA within a highly complex sample background to determine carnitine monooxygenase inhibitory potential of natural extracts.

The human gut microbiome plays a key role in human health . The advent of the omics-based technologies has opened a new scenario in the comprehension of the gut ecosystem. In particular, the study of metabolites related to the gut microbiota can offer deep insights into gut microbial functional capacity and its impact on the host in health and disease. In this context, Metabolomics has become a powerful approach to increase our understanding of potentially important metabolites that derive from the gut microbiota that may impact host physiology. Metabolomics is con cerned with the study of the global changes in the metabolites present within biological systems. lt has matured to a frequent available technology in many fields of application, largely driven by improved mass spectrometry with increased sensitivity and metabolite coverage. This keynote aims at providing an introduction to the metabolomics workflow for studying complex interrelationships between intestinal microbiota and their human host, with particular focus on strengths and weaknesses of using mass spectrometry-based analytical platforms. In this context, recent knowledge in the understanding o( the role of gut microbiota as an active participant in the development of atherosclerosis and its adverse complications of cardiovascular disease will be summarized.

[Objectives]: Elevated levels of trimethylamine N-oxide (TMAO) in plasma has been recently proposed as a risk factor in cardiovascular disease and has shown to have prognostic value in various studies. TMAO is generated via a metaorganismal pathway that starts with conversion of dietary precursors (choline, L-carnitine, etc.) into trimethylamine (TMA) by gut microbiota, followed by oxidation by host liver enzymes. The discovery of novel dietary compounds able to interfere with the microbial TMA formation has attracted much attention in recent years. However, to date there have been no reports of dietary compounds or natural extracts able to inhibit the activity of carnitine monooxygenase, a microbial enzyme that features the conversion of L-carnitine to TMA. The main goal of the present work was the development of a methodology that combines resazurin-based microplate assay with a CE-UV method for the determination of carnitine monooxygenase inhibitory potential of natural extracts., [Methods]: In this work, a TMA producer strain of Klebsiella pneumoniae, isolated from human gut, was used. The fluorescent signal derived from bacterial growth was measured using Synergy HT microplate reader (BioTek Instruments), and TMA accumulation in bacterial samples was derivatized with 2,4′-dibromoacetophenone reagent and analyzed using a P/ACE 5010 CE system equipped with a diode array UV-Vis detector (Beckman Coulter)., [Results]: The analytical methodology was based on the combined use of a microdilution method with resazurin reagent for real-time monitoring of bacterial viability and a CE-UV method to determine the accumulation of TMA in samples. During the optimization of the methodology, different parameters affecting the fluorescence signal and CE analysis were investigated. The optimized conditions were successfully applied to assay the carnitine monooxygenase inhibitory potential of a collection of natural extracts., [Conclusions]: The proposed methodology that combines a microplate fluorescence assay and CE-UV to simultaneously monitor the bacterial growth and metabolic rate for TMA production has shown to be a suitable procedure to determine carnitine monooxygenase inhibitory potential of natural extracts.