Neuroactive compounds in Tomatoes and their role in the development of neurodegenerative diseases via the gut-brain axis using a multi-Omics approach

Project Information

NeuroTOm

Grant agreement ID: 101062798

DOI

10.3030/101062798

Project closed

EC signature date 9 August 2022

Start date 1 November 2022

End date 31 October 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 172 750,08

Coordinated by

FONDAZIONE EDMUND MACH
Italy

Periodic Reporting for period 1 - NeuroTOm (Neuroactive compounds in Tomatoes and their role in the development of neurodegenerative diseases via the gut-brain axis using a multi-Omics approach)

Reporting period: 2022-11-01 to 2024-10-31

Summary of the context and overall objectives of the project

Despite growing evidence of a connection between diet, the gut microbiome, and neurodegenerative diseases, the exact relationship remains unclear. The NeuroTOm project aims to deepen our understanding of the diet-gut microbiome-brain axis and contribute to the prevention of these diseases. Using tomatoes as a model food, the project applied state-of-the-art targeted and non-targeted analytical methods (LC-MS/MS, GC-MS/MS) to identify neuroactive compounds in tomatoes and investigate the fate of these compounds during digestion, fermentation, and their impact on the gut microbiome using in vitro digestion and colon models.
Neurodegenerative diseases are debilitating conditions affecting millions worldwide, including over 7 million Europeans. Although the etiology of these disease is complex, growing evidence suggests that dietary factors may influence their progression. Tomatoes, a widely consumed vegetable, are rich in bioactive compounds like phenolics, carotenoids, and glycoalkaloids, which show potential neuroprotective properties. However, most research has focused on specific nutrients, often overlooking neuro-disrupting compounds in tomato-based products, such as mycotoxins, pesticides, and industrial chemical residues. This raises concerns about the overall nutritional benefits and the impact of these contaminants on the gut microbiome and brain health. Moreover, many compounds remain unidentified, complicating efforts to link dietary exposure to human health outcomes. To address this knowledge gap, the objective of our project was to develop advanced analytical techniques to characterize both beneficial and harmful neuroactive compounds in tomato-based products, providing a better understanding of human exposure through food digestion.
Understanding the biological effects of neuroactive compounds requires investigating what happens to these compounds after digestion. Bioavailability depends on factors like digestive stability, release from the food matrix, metabolic behavior, and trans-epithelial passage. However, there is limited knowledge about the in vitro gastrointestinal digestion of tomato-based products. Existing studies often focus on individual neuroprotective compounds, neglecting the interactions between these and neuro-disrupting substances. It remains unclear how neuro-disrupting compounds may influence the fate of neuroprotective compounds and their effect on the gut microbiome during digestion. Within the scope of this project, we aimed to increase knowledge about the fate of neuroactive compounds once they enter the gastrointestinal tract and how this affects the gut microbiome.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project developed advanced multi-omics methodologies to identify neuroprotective and neuro-disrupting compounds in tomatoes, including their metabolites before and after digestion, as well as in fecal samples. The research aimed to evaluate the neuroactive profile of tomatoes, investigate the fate of these compounds once digested, and assess their impact on the human gut microbiome. The first experiment focused on profiling neuroactive compounds in four tomato types: organic and conventional datterini tomatoes, conventional plump tomatoes, and processed tomatoes. Principal Component Analysis revealed that the major differences in compound profiles were between the different processing methods and types of tomatoes, rather than between production methods (organic vs. conventional). Over 70 compounds were identified, including neuroprotective (e.g. polyphenols, amino acids, neurotransmitters) and neuro-disrupting compounds (e.g. pharmaceuticals, food additives). The annotation strategy also helped identify markers specific to certain conditions, such as markers of organic production, variety, and process, as well as non-group-specific neuroactive compounds. Processed tomatoes contained the highest number of neuroactive compounds, which did not suggest lower food quality, nor was higher quality observed for organically produced tomatoes. Additionally, smaller varieties were found to be richer in terms of nutritional value. The carotenoid distribution analysis suggested that the majority of carotenoids were concentrated in the tomato peel rather than the fruit body. While most compounds were neuroprotective, our study shows that their presence was accompanied by neuro-disrupting compounds.
In vitro-simulated gastrointestinal digestion and colon fermentation models were used to study the fate of neuroactive compounds in the gut microbiome. The kinetics of different compounds during digestion and fermentation focused on bioaccessibility, bioavailability, biotransformation, and excretion patterns. Key findings include:
- Some compounds showed a preference for excretion via urine.
- Others reached the colon, and a few demonstrated potential for crossing the blood-brain barrier, including certain neuro-disrupting compounds.
- The presence of tomato or neuro-disrupting compounds influenced the behaviour and bioavailability of some compounds.
Fermentation revealed differences between samples with and without digested tomatoes, as well as between tomato samples with and without neuro-disrupting compounds. However, the number and amount of neuroprotective compounds were higher when tomato was present in the fermentation sample.
The project developed a robust non-targeted analysis workflow using mass spectrometry and statistical methods for filtering mass features. Tools such as MZmine, Sirius, and GNPS were employed to tentatively identify unknown compounds (e.g. fructosyl pyroglutamate, bile acids). The analysis of short-chain fatty acids, and metagenomics revealed effects on the gut microbiome. Although no significant changes in reactive oxygen species levels were found, short-chain fatty acids and bile acids formation were influenced by the presence of neuroactive compounds.
The project has been experimentally completed, and data has been processed and discussed. Some preliminary results were presented at two research conferences. The full findings are expected to be published in two papers by January 2025, with at least two more papers expected by May 2025. All R-codes, raw and processed data, and publications will be made publicly available in trusted repositories (Zenodo, GitHub) once the papers are published.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The conducted research has advanced beyond the previous state-of-the-art by developing novel analytical methods and workflows (both targeted and non-targeted analysis) for monitoring diverse compounds, including neuroprotective and neuro-disrupting compounds and their gut-microbiome metabolites, in food and cell extracts. Unlike most studies that focus on individual compounds or processes without considering prior processing steps, this project offers a more comprehensive approach by following the realistic pathway of compounds entering the human body through food. The study design performed enabled to evaluate the effect of digestion on neuroactive compounds and, therefore, their potential of bioaccessibility and bioavailability while reaching the microbiome. Due to the interdisciplinary nature of the project, it provides valuable insights into the behavior and effects of these compounds on the human microbiome and their potential role in the gut-brain axis.
The findings demonstrate the dynamic nature of neuroactive compound formation and transformation during digestion and fermentation. Importantly, while confirming the richness of tomato products in terms of neuroactive compounds, the project goes beyond the state of the art by identifying the presence of neurotoxic compounds. It also provides information on the potential for these compounds to reach the colon and, potentially, the brain, and their role in the development of neurodegenerative diseases.
Additionally, the project received co-funding through reintegration to expand the NeuroTOm project. This phase will apply the research to other food products, continue using 2D in vitro models, and incorporate 3D models like brain and intestinal organoids. The next goal is to correlate the metabolomics data with clinical interventional studies, planned for future collaboration with clinical institutions.

The project's experimental design