Serum Metabolites Responding in a Dose-Dependent Manner to the Intake of a High-Fat Meal in Normal Weight Healthy Men Are Associated with Obesity

Although the composition of the human blood metabolome is influenced both by the health status of the organism and its dietary behavior, the interaction between these two factors has been poorly characterized. This study makes use of a previously published randomized controlled crossover acute intervention to investigate whether the blood metabolome of 15 healthy normal weight (NW) and 17 obese (OB) men having ingested three doses (500, 1000, 1500 kcal) of a high-fat (HF) meal can be used to identify metabolites differentiating these two groups. Among the 1024 features showing a postprandial response, measured between 0 h and 6 h, in the NW group, 135 were dose-dependent. Among these 135 features, 52 had fasting values that were significantly different between NW and OB men, and, strikingly, they were all significantly higher in OB men. A subset of the 52 features was identified as amino acids (e.g., branched-chain amino acids) and amino acid derivatives. As the fasting concentration of most of these metabolites has already been associated with metabolic dysfunction, we propose that challenging normal weight healthy subjects with increasing caloric doses of test meals might allow for the identification of new fasting markers associated with obesity

The NutriChip project - translating technology into nutritional knowledge

Advances in food transformation have dramatically increased the diversity of products on the market and, consequently, exposed consumers to a complex spectrum of bioactive nutrients whose potential risks and benefits have mostly not been confidently demonstrated. Therefore, tools are needed to efficiently screen products for selected physiological properties before they enter the market. NutriChip is an interdisciplinary modular project funded by the Swiss programme Nano-Tera, which groups scientists from several areas of research with the aim of developing analytical strategies that will enable functional screening of foods. The project focuses on postprandial inflammatory stress, which potentially contributes to the development of chronic inflammatory diseases. The first module of the NutriChip project is composed of three in vitro biochemical steps that mimic the digestion process, intestinal absorption, and subsequent modulation of immune cells by the bioavailable nutrients. The second module is a miniaturised form of the first module (gut-on-a-chip) that integrates a microfluidic-based cell co-culture system and super-resolution imaging technologies to provide a physiologically relevant fluid flow environment and allows sensitive real-time analysis of the products screened in vitro. The third module aims at validating the in vitro screening model by assessing the nutritional properties of selected food products in humans. Because of the immunomodulatory properties of milk as well as its amenability to technological transformation, dairy products have been selected as model foods. The NutriChip project reflects the opening of food and nutrition sciences to state-of-the-art technologies, a key step in the translation of transdisciplinary knowledge into nutritional advic

From food to physiology characterization and in vitro digestion of milk products and investigation of postprandial metabolism and inflammation after a high-fat meal intake in a human nutrition intervention study

According to the World Health Organization worldwide obesity has more than doubled since 1980. The World health statistics 2012 report shows that one in six adults is obese, one in ten diabetic and one in three has a raised blood pressure. Overweight and obesity are major risk factors for cardiovascular diseases,According to the World Health Organization worldwide obesity has more than doubled since 1980. The World health statistics 2012 report shows that one in six adults is obese, one in ten diabetic and one in three has a raised blood pressure. Overweight and obesity are major risk factors for cardiovascular diseases, diabetes and some cancers. The causes of overweight and obesity are mainly a decrease in physical activity and an increased intake of energy-dense foods leading to a positive energy balance. Not only dietary patterns play a major role in the development of obesity. Food composition also directly impacts on postprandial metabolism and inflammation and, in long term, may contribute to systemic low-grade inflammation, a characteristic associated with the obese state. Thus it is not surprising that there is a growing interest in better understanding the effect of different foods and food compounds on human metabolism and health. The aim of the NutriChip project is to develop a microfluidic chip device to screen foods and their compounds for health promoting, e.g. immune-modulatory, properties. Milk products have been chosen as a food model because their consumption has been shown to be associated with decreased levels of inflammatory markers (1), and also because technological and microbiological transformations allow the realization of various products, such as yoghurt and cheese, having potentially different physiological effects. This thesis covers the biochemical (products characterization and in vitro digestion) and physiological (human nutrition study) aspects of the NutriChip project. Initially, differently heat-treated and fermented milk products were characterized by a proteomic approach. Various fractionation methods were used for selectively enriching minor milk and bacterial proteins in different dairy products. Proteins were separated and identified by two dimensional (2D) gel-electrophoresis and liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. All data are collected in an interactive platform, called protein atlas, which is now publicly available on http://www.foodle.ch/de/proteinstart. Currently, the database contains more than 200 different milk proteins and about 250 bacterial proteins. For a better in depth characterization of fermented products, a method for the enrichment of living bacterial cells has been developed. This method allows the investigation of bacterial proteomes under different fermentation conditions. The method was used to monitor the adaption of the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus during milk fermentation. Some well-known stress response proteins (chaperone proteins GroEL and DnaK) and various enzymes involved in the glycolysis pathway have been identified as a proof of concept. Another experiment aimed to investigate the bacterial proteome during cheese production with special regards towards the identification of enzymes involved in the formation of the cheese flavor compound 3-methylbutanal. An aminotransferase that catalyzes the first step in the conversion pathway from the amino acid leucine to the flavor compound 3-methylbutanal could be identified. A prerequisite for screening foods for health-modulating activity was the development of a static, three-step in vitro digestion model. The aim was to keep the model as physiological as possible and ideally perform the digestion on a small scale (volume < 15 mL). The digestion model has been thoroughly validated using pasteurized whole milk. The degradation of fat, carbohydrates and proteins into their basic constituents was consistent with human physiological values found in the literature. The system has been used to digest various milk products. With the two proteins α-s1-casein and β-lactoglobulin, a representative of the caseins and whey proteins, respectively, the influence of the fat content, heat treatment and fermentation of the products on their digestibility has been monitored. Particularly we also looked at the generation of bioactive peptides during the digestion process. Towards this aim, the identified peptides in the digestion experiments were compared with bioactive peptide sequences from the literature using the statistic program R. Over 50 milk protein-derived peptides containing bioactive peptide sequences, with e.g. antihypertensive, immune- and cyto-modulatory, opioid functions, could be identified. The next step in simulating the human digestive system is the intestinal transport. Therefore, a cell culture model mimicking the last step of digestion and final absorption of the nutrients, both steps being mediated by intestinal enterocytes, was established. For this aim, Caco-2 cells were used in a Transwell system with an upper and lower compartment containing cell culture medium. Digested milk products were added on top of the Caco-2 cell monolayer and medium was collected from the basolateral chamber for identification of transported, potential bioactive peptides. The final validation of the in vitro models needs the comparison with a human nutrition intervention study. We designed and conducted a dose-response intervention study to determine the caloric dose (500 kcal, 1’000 kcal, 1’500 kcal) of a high-fat meal needed to induce a postprandial metabolic and inflammatory response in normal weight and obese subjects. In our study, we investigated postprandial metabolism and inflammation by measuring classical clinical parameters such as glucose, insulin and triglycerides, as well as the inflammation markers C-reactive protein (CRP), interleukine-6 (IL-6) and endotoxin before and at various time points after the test meals consumption. Our study provided valuable clinical, mechanistic, and methodological insights into the metabolic response of subjects, varying in their metabolic health status, to increasing doses of a high-fat meal. This represents the basis for future studies aiming to investigate health-promoting properties of foods in general, as also their capability in lowering postprandial inflammation, a normal response mechanism after food ingestion. v diabetes and some cancers. The causes of overweight and obesity are mainly a decrease in physical activity and an increased intake of energy-dense foods leading to a positive energy balance. Not only dietary patterns play a major role in the development of obesity. Food composition also directly impacts on postprandial metabolism and inflammation and, in long term, may contribute to systemic low-grade inflammation, a characteristic associated with the obese state. Thus it is not surprising that there is a growing interest in better understanding the effect of different foods and food compounds on human metabolism and health. The aim of the NutriChip project is to develop a microfluidic chip device to screen foods and their compounds for health promoting, e.g. immune-modulatory, properties. Milk products have been chosen as a food model because their consumption has been shown to be associated with decreased levels of inflammatory markers (1), and also because technological and microbiological transformations allow the realization of various products, such as yoghurt and cheese, having potentially different physiological effects. This thesis covers the biochemical (products characterization and in vitro digestion) and physiological (human nutrition study) aspects of the NutriChip project. Initially, differently heat-treated and fermented milk products were characterized by a proteomic approach. Various fractionation methods were used for selectively enriching minor milk and bacterial proteins in different dairy products. Proteins were separated and identified by two dimensional (2D) gel-electrophoresis and liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis. All data are collected in an interactive platform, called protein atlas, which is now publicly available on http://www.foodle.ch/de/proteinstart. Currently, the database contains more than 200 different milk proteins and about 250 bacterial proteins. For a better in depth characterization of fermented products, a method for the enrichment of living bacterial cells has been developed. This method allows the investigation of bacterial proteomes under different fermentation conditions. The method was used to monitor the adaption of the yoghurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus during milk fermentation. Some well- known stress response proteins (chaperone proteins GroEL and DnaK) and various enzymes involved in the glycolysis pathway have been identified as a proof of concept. Another experiment aimed to investigate the bacterial proteome during cheese production with special regards towards the identification of enzymes involved in the formation of the cheese flavor compound 3-methylbutanal. An aminotransferase that catalyzes the first step in the conversion pathway from the amino acid leucine to the flavor compound 3-methylbutanal could be identified. v A prerequisite for screening foods for health-modulating activity was the development of a static, three-step in vitro digestion model. The aim was to keep the model as physiological as possible and ideally perform the digestion on a small scale (volume < 15 mL). The digestion model has been thoroughly validated using pasteurized whole milk. The degradation of fat, carbohydrates and proteins into their basic constituents was consistent with human physiological values found in the literature. The system has been used to digest various milk products. With the two proteins α-s1-casein and β-lactoglobulin, a representative of the caseins and whey proteins, respectively, the influence of the fat content, heat treatment and fermentation of the products on their digestibility has been monitored. Particularly we also looked at the generation of bioactive peptides during the digestion process. Towards this aim, the identified peptides in the digestion experiments were compared with bioactive peptide sequences from the literature using the statistic program R. Over 50 milk protein-derived peptides containing bioactive peptide sequences, with e.g. antihypertensive, immune- and cyto-modulatory, opioid functions, could be identified. The next step in simulating the human digestive system is the intestinal transport. Therefore, a cell culture model mimicking the last step of digestion and final absorption of the nutrients, both steps being mediated by intestinal enterocytes, was established. For this aim, Caco-2 cells were used in a Transwell system with an upper and lower compartment containing cell culture medium. Digested milk products were added on top of the Caco-2 cell monolayer and medium was collected from the basolateral chamber for identification of transported, potential bioactive peptides. The final validation of the in vitro models needs the comparison with a human nutrition intervention study. We designed and conducted a dose-response intervention study to determine the caloric dose (500 kcal, 1’000 kcal, 1’500 kcal) of a high-fat meal needed to induce a postprandial metabolic and inflammatory response in normal weight and obese subjects. In our study, we investigated postprandial metabolism and inflammation by measuring classical clinical parameters such as glucose, insulin and triglycerides, as well as the inflammation markers C-reactive protein (CRP), interleukine-6 (IL-6) and endotoxin before and at various time points after the test meals consumption. Our study provided valuable clinical, mechanistic, and methodological insights into the metabolic response of subjects, varying in their metabolic health status, to increasing doses of a high-fat meal. This represents the basis for future studies aiming to investigate health- promoting properties of foods in general, as also their capability in lowering postprandial inflammation, a normal response mechanism after food ingestion

Impact of milk processing on the generation of peptides during digestion

Milk processing may induce changes in dairy product composition and influence digestibility and nutrient bioavailability. Differences in protein degradation and peptide generation were studied for β-lactoglobulin and αS1-casein from commercially available dairy products before, during, and after in vitro digestion. All major milk proteins, except β-lactoglobulin, were degraded to smaller peptides during the gastric phase in all investigated products. After the gastric phase, a shortened fragment of β-lactoglobulin was identified in the non-fermented dairy products, underlining differences in protein conformation due to the fermentation process. During the gastric phase, greater numbers of small peptides were generated from αS1-casein than from β-lactoglobulin. The monitoring of generation of specific β-lactoglobulin and αS1-casein peptide profiles by liquid chromatography–mass spectrometry allowed the identification of potential bioactive peptides. Peptides with satiety-influencing DPP-4 inhibiting properties were monitored and quantities were compared between products to identify promising targets for the development of new health promoting products

Nutrichip: an integrated microfluidic system for in vitro investigation of the immunemodulatory function of dairy products

We introduce the concept of the NutriChip, a miniaturized artificial human gastrointestinal tract (GIT) for investigating the potential of immuno-modulatory function of dairy food. More specifically, we are investigating the effect of digested milk on immune cells activation by creating conditions close to those of the human GIT employing an in vitro model of hu-man GIT epithelial cells (Caco-2) in co-culture with human immune cells (differentiated THP-1). The cytokines (IL1/6) and Toll-like receptors (TLR-2/4) expressions by the immune cells will be monitored upon the application of dairy food on the epithelial cell layer

The NutriChip project--translating technology into nutritional knowledge

Advances in food transformation have dramatically increased the diversity of products on the market and, consequently, exposed consumers to a complex spectrum of bioactive nutrients whose potential risks and benefits have mostly not been confidently demonstrated. Therefore, tools are needed to efficiently screen products for selected physiological properties before they enter the market. NutriChip is an interdisciplinary modular project funded by the Swiss programme Nano-Tera, which groups scientists from several areas of research with the aim of developing analytical strategies that will enable functional screening of foods. The project focuses on postprandial inflammatory stress, which potentially contributes to the development of chronic inflammatory diseases. The first module of the NutriChip project is composed of three in vitro biochemical steps that mimic the digestion process, intestinal absorption, and subsequent modulation of immune cells by the bioavailable nutrients. The second module is a miniaturised form of the first module (gut-on-a-chip) that integrates a microfluidic-based cell co-culture system and super-resolution imaging technologies to provide a physiologically relevant fluid flow environment and allows sensitive real-time analysis of the products screened in vitro. The third module aims at validating the in vitro screening model by assessing the nutritional properties of selected food products in humans. Because of the immunomodulatory properties of milk as well as its amenability to technological transformation, dairy products have been selected as model foods. The NutriChip project reflects the opening of food and nutrition sciences to state-of-the-art technologies, a key step in the translation of transdisciplinary knowledge into nutritional advice

Validation of an in vitro digestive system for studying macronutrient decomposition in humans

The digestive process transforms nutrients and bioactive compounds contained in food to physiologically active compounds. In vitro digestion systems have proven to be powerful tools for understanding and monitoring the complex transformation processes that take place during digestion. Moreover, the investigation of the physiological effects of certain nutrients demands an in vitro digestive process that is close to human physiology. In this study, human digestion was simulated with a 3-step in vitro process that was validated in depth by choosing pasteurized milk as an example of a complex food matrix. The evolution and decomposition of the macronutrients was followed over the entire digestive process to the level of intestinal enterocyte action, using protein and peptide analysis by SDS-PAGE, reversed-phase HPLC, size exclusion HPLC, and liquid chromatography-MS. The mean peptide size after in vitro digestion of pasteurized milk was 5-6 amino acids (AA). Interestingly, mostly essential AA (93.6%) were released during in vitro milk digestion, a significantly different relative distribution compared to the total essential AA concentration of bovine milk (44.5%). All TG were degraded to FFA and monoacylglycerols. Herein, we present a human in vitro digestion model validated for its ability to degrade the macronutrients of dairy products comparable to physiological ranges. It is suited to be used in combination with a human intestinal cell culture system, allowing ex vivo bioavailability measurements and assessment of the bioactive properties of food components