In Phase 1, this research focused on how compounds in tomatoes, specifically polyphenols and carotenoids, interact with starch at a molecular level. The aim was to determine whether these bioactive compounds could reduce the digestibility of starch, potentially mitigating the sharp rise in blood sugar typically experienced after consuming starchy foods. A single-enzyme digestion system, simulating starch breakdown in the human digestive process, was used to explore this interaction and its potential effects on blood sugar responses.
Selection of Tomato Varieties
Three tomato types were selected for their unique bioactive profiles, providing insight into the optimal source of polyphenols and carotenoids:
MITOS Tomato Sauce: Used in the MITOS clinical study examining the effects of long-term tomato intake on metabolic health, this source offered a unique opportunity to align in vitro-based results from this research project with the outcomes from the MITOS ongoing dietary intervention, which samples and data will be used to achieve objective 3 in this project.
Blue Tomatoes (cultivar Mar Azul): Known for their high anthocyanin levels, blue tomatoes offer an ideal source of these bioactive compounds, which are thought to affect starch breakdown by digestive enzymes.
Pebroter Tomatoes (cultivar Corno Andino): Locally grown in Catalonia and harvested in September 2024, these tomatoes are rich in polyphenols. Previous studies have shown that this variety has a consistently high polyphenol content and promising yield characteristics, making it a strong candidate for this research project.
Extraction and Analysis of Bioactive Compounds
To extract polyphenols and carotenoids, a green solvent extraction method using methanol and ethanol was employed. Each tomato source underwent specific preparation to ensure accurate fractionation:
• Blue and Pebroter Tomatoes: These raw tomatoes were washed, manually peeled, and deseeded, allowing for the separation and analysis of bioactive compounds in the peel, seeds, and pulp.
• Tomaro paste: As this sauce was industrially processed, it was not possible to separate fractions; instead, the entire sauce was analysed.
Following extraction, liquid chromatography–mass spectrometry (LC-MS) was used to identify and quantify polyphenols (currently ongoing), while the total polyphenol content was determined using the Folin-Ciocalteu method. Extracts were tested in various concentrations with wheat and rice starch to determine the concentration required for an effect on starch digestibility and whether starch type significantly impacted digestion profiles.
Carotenoids were extracted and will be quantified to be later used for in vitro digestions.
Starch Digestibility Assay and Quantification of Reducing Sugars
The starch digestibility assay applied here was previously validated to correlate well with glycaemic index (GI) values obtained in vivo. Digestibility was measured by assessing the reducing sugars released during starch breakdown, analysed through colorimetric methods based on p-hydroxybenzoic acid hydrazide (PAHBAH) and dinitrosalicylic acid (DNS) assays. Due to potential UV/visible absorbance interference from polyphenols in the extracts, a high-performance liquid chromatography (HPLC) coupled with Refractive Index detector method was also developed to accurately quantify polysaccharides and ensure reliable digestion data.
Phenolic and carotenoids copounds were identified and quantified using LC-MS/MS and UPLC-DAD, respectively.