Preliminary results

Preliminary experiments of digestion of tomato:

• Neuroactive compounds present at low concentrations were adjusted prior to digestion.

• Neuroactive compounds were analyzed in separate digestion phases during digestion and after:

  • oral, gastro, and intestinal phases during digestion,

  • liquid and pallet after digestion.

• Based on the obtained results the possible selection of neuroactive compounds is as follows: 

  • neuroprotective compounds: rutin, chlorogenic acid, b-carotene, eriodictyol,

  • neuro-disrupting compound: aspartame, tomatine, bisphenol S, difenoconazole, acetaminophen.

• Results suggest that some compounds, e.g., phenylalanine and, tryptone are highly present in the digested matrix and are also forming during digestion, and although present at high concentrations not exactly tomato specific.

• Compounds with a preferable presence in the liquid phase after digestion are more comment to be extracted with urine while neuroactive compounds present in pallets have a higher potential to reach the human microbiome-colon. 

Digestion of tomato - testing variabilty of tomato digestion experiemnt:

• Neuroactive compounds present at low concentrations were adjusted prior to digestion.

• Neuroactive compounds were analyzed in separate digestion phases during digestion and after:

  • oral, gastro, and intestinal phases during digestion,

  • liquid and pallet after digestion.

• Based on the obtained results the final selection of neuroactive compounds is as followed:

  • neuroprotective compounds: rutin, tomatine, pantotheic acid

  • neuro-disrupting compound: aspartame, bisphenol S, difenoconazole. 

  • the selection was based also on different kinetic profiles from preliminary fecal experiemnt, namely the aim was selected compounds reaching the microbiome in which they also react differently. 

• Results suggest that some compounds, e.g., phenylalanine and, tryptone are highly present in the digested matrix and are also forming during digestion, and although present at high concentrations not exactly tomato specific.

• The results confirmed the repeatabilty of the experiment - the concentration of majorioty of neuroactive compound were < 20% in digested tomato pallet confirming the digestion model repeatabilty. 

Preliminary experiments of fecal fermentation experiment with digested material of tomato:

• The experiment included two conditions, namely blank with fecal only and sample with fecal and digested tomato which was prior digestion adjusted with the neuroactive compounds of interest. 

• Time points 0, 10, and 24 h to monitor the degradation kinetic and biotransformation of neuroactive compounds during fecal fermentation

• We tested different sample preparations for all the analyses planned for the final experiment: metabolomics, ROS, SCFA, and metagenomic analysis 

  • metabolomics: optimal sample preparation using SPE Oasis Prime HLB 30mg 96well plates, 10x concentration of sample extract (although we lose some neuroactive compounds, however, they are less important in terms of this WP aim),

  • SCFA, following Lotti et al. (2017),

  • metagenomic, DNA extracts prepared by following the manufacturer's recommendation, QIAamp PowerFecal DNA Kit (QIAGEN, Germany). 

Target analysis of digestion and fecal fermentation model selected withinh this project with the aim to answear the project's aim:

• Tomato was digested following infogest protocol withouth and with the addition of neuro-disrupting compounds at the highest tolerabily intake of BPS as the most restricted compound among three selected.

• The experiemnt consisted of 10+2 fecal vessels, positive control - inulin, digested tomato with and withouth addition of neurodisrupting compunds, fecal vessels with the addition of rutin, tomatine, pantotheic acid, aspartame, difenoconazole and bisphenol S. 

• The preliminary results confirmed the appropriatness of experimental design. 

  • there is a difference between adjusted and non-adjusted tomato,

  • the biotransformation difference between differenet neuroactive compounds,

  • the signal of selected compounds is good at time zero.