Dietary Proteins: Metagenomic and Metabolomics Approaches for Human Biomarkers Identification

Difference in faecal metabolites depending on the protein supplementation amount and from colonic in vitro fermentation. Targeted and untargeted metabolomics measurements will be used (e.g., short-chain fatty acids and branched chain fatty acids analyses, and polar and non polar metabolomics). Faecal samples will have their metabolites extracted and quantified by liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-qToF-MS), and gas chromatography coupled with high-resolution mass spectroscopy (GC-Orbitrap). The level of metabolites will be compared throughout the whole study.

Difference in plasma metabolites (blood obtained fastened state) depending on the protein supplementation amount. Targeted and untargeted metabolomics measurements will be used (e.g., lipids, and polar and non polar metabolomics). Plasma samples will have their metabolites extracted and quantified by liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-qToF-MS), and gas chromatography coupled with high-resolution mass spectroscopy (GC-Orbitrap). The level of metabolites will be compared throughout the whole study.

Difference in the gut microbiota profile/composition because of the protein supplementation and from colonic in vitro fermentation will be assessed by 16s/NGS. Alpha and Beta diversity will be measured and compared throughout the 4 weeks of intervention and baselines. In the case of NGS, the genome sequence will be annotated and the resulting set of genes encoding for metabolic enzymes will be extracted. Data from shotgun metagenomics and metabolomics will be integrated by constructing dependency networks, with a special attempt to infer causal relationships among different variables.

Difference in 24 h urine samples markers due to the protein supplementation (e.g., for 24 h urine: urea nitrogen from 24 h urine, creatinine, uric acid)

Difference in blood (serum) samples markers due to the protein supplementation (e.g., urea and creatinine)

Difference in plasma metabolites depending on the protein taken as breaskfast. Targeted and untargeted metabolomics measurements will be used (e.g., polar and non polar metabolomics). Plasma samples will have their metabolites extracted and quantified by liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-qToF-MS), and gas chromatography coupled with high-resolution mass spectroscopy (GC-Orbitrap). The level of metabolites will be compared with those found in the fastening state.

Subjects will have their fresh faecal samples used in the colonic in vitro fermentation experiment to evaluate whether and how gut metabolites production can be predicted in vitro. The same proteins used in the dietary intervention study will be digested in vitro and used for the batch in vitro colonic fermentation. Supernatant will be used for metabolomics analysis (outcome 1) and selected fermentation time point pellets will be used for microbiota composition and functional analyses (outcome 3).

Food diaries 3 times a week.

The gastrointestinal symptoms will be assessed during the baseline and protein supplementation period. Difference in the frequency and severity of gastrointestinal symptoms during the study will be assessed (15 questions with a scale of 1-7, minimum value 1, maximum 7, higher score correspond to a worse outcome)

The bowel movement will be assessed during the baseline and protein supplementation period by the Bristol scale diary. In the diary they write down the type of stool based on the scale which shows 7 pictures of different forms of stool, from watery diarrhea to compact. Participants are asked to choose the form of stool they have every day during the study.

To assess the maintenance or difference of physical activity level during the baseline and dietary intervention by the AKTIVITETSVANOR questionnaire (Swedish questionnaire to assess physical activity frequency for 7 days).

The height will be assessed in meters.

Participants will be weighted in order to have information of their body weight changes in kilograms during the study.

Participants will be have their body composition measured using Tanita® full body scale. The obtained results by Tanita are: weight (kg), muscle mass (%) and kg), body water content (% and kg), fat mass (% and kg), basal energy expenditure (kcal and kJ) and BMI in kg/m^2.

Difference in faecal levels of calprotectin during the study intervention

Measurement of glucose in blood samples

Measurement of insulin in blood samples

Measurement of C-reactive protein in blood samples

Measurement of cholesterol (total, LDL, and HDL) in blood samples (fasting state)

Measurement of triglycerides in blood samples (fasting state)

Measurement of glucose in blood samples after the dietary challenge to check its level, compare with the fastening state and evaluate the area under the curve.

Measurement of insulin in blood samples after the dietary challenge to check its level, compare with the fastening state and evaluate the area under the curve.

Difference in the gut microbiota profile/composition 1 and 2 after the protein supplementation will be assessed by 16s/NGS. Alpha and Beta diversity will be measured and compared throughout these 2 weeks after the intervention. In the case of NGS, the genome sequence will be annotated and the resulting set of genes encoding for metabolic enzymes will be extracted. Data from shotgun metagenomics and metabolomics will be integrated by constructing dependency networks, with a special attempt to infer causal relationships among different variables.

Difference in faecal metabolites levels after the intervention will be measured. Targeted and untargeted metabolomics measurements will be used (e.g., short-chain fatty acids and branched chain fatty acids analyses, and polar and non polar metabolomics). Faecal samples will have their metabolites extracted and quantified by liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-qToF-MS), and gas chromatography coupled with high-resolution mass spectroscopy (GC-Orbitrap). The level of metabolites will be compared after 1 and 2 weeks of taking the protein supplements.

From the subset of the participants, a breath sample will be collected by having them breathe into a breath sample collecting device (ReCIVA, Owlstone, UK), in order to measure volatile organic compounds by thermal desorption-gas chromatography coupled to ultra-resolution mass spectrometry (TD-GC-HRMS-Orbitrap).