Final Report Summary - GIST (Gastro-intestinal behaviour of phytosterols and enzymatic modification thereof) Introduction Phytosterols are added to foods as a functional food ingredient because of their cholesterol lowering capacity. Even though the exact mechanism is still unclear, phytosterols prevent the uptake of cholesterol in the small intestine and are not absorbed themselves. So, the phytosterols end up in the colon where they might be metabolised by the human gut microbiota. The aim of this research was to study if the phytosterols are metabolised by the human gut microbiota, what metabolites are being formed and if incubation with phytosterols affect the growth characteristics of the gut microbiota. The research has been dived into two parts. In the first part, the analytical method needed to be able to extract and detect the phytosterols from fermentation matrices was developed and validated. In the second part, phytosterols were incubated with a bacterial suspension representing the human intestinal ecosystem to be able to study the metabolism of phytosterols and the effect thereof on the growth characteristics of the gut bacteria. Results A new ultrahigh-performance liquid chromatography - atmospheric pressure photoionisation - mass spectrometry (UHPLC-APPI-MS) method was developed for the detection and identification of the phytosterols and its potential metabolites. Phytosterols are separated on a Waters UHPLC BEH C18 column using a gradient from 50 % methanol in water to 100 % methanol. The separation method was designed based on standard compounds. In the final method, three important phytosterols and cholesterol could be separated and enough separation space was available for the potential metabolites. Solid phase extraction (SPE) was used for the extraction of phytosterols from fermentation matrices. Extraction by liquid-liquid extraction was found to give highly variable recoveries, while extraction by SPE was found to give recoveries of 70 - 80 %. The phytosterols were eluted from the SPE cartridges by isopropanol. As a result of that, the choice of cartridge appeared to be important. The combined method of SPE extraction and UHPLC-MS analysis was validated and a proof of concept was given by studying the stability of phytosterols under stomach and small intestinal conditions. The phytosterols stigmasterol, stigmastanol and sitostanol and cholesterol were found to be stable under the conditions of the stomach and small intestine as expected based on literature. Secondly, one of the often studied phytosterols, stigmasterol, was incubated with a bacterial slurry representing the human microbial ecosystem. Stigmasterol was incubated at two concentration levels for 72 hours at 37 degrees of Celsius and at several time points samples were taken. These samples were analysed for metabolites using the method developed in the first part of this study and for short chain fatty acid (SCFA) formation. Furthermore, the growth and the pH were monitored. The bacterial counts and the pH were found to be stable during the whole incubation period. However, SCFA were formed during the 72 h of incubation, showing that the bacteria were metabolically active. It was remarkable that the amount of SCFA produced was concentration dependent. The amount SCFA produced by the blank was significantly higher than in the incubations containing phytosterols. This could indicate a negative influence of stigmasterol on the microbial metabolism. The analysis of the samples after incubation by UHPLC-MS showed that the recovery of stigmasterol was similar in the incubations containing stigmasterol and microbiota and incubations containing only stigmasterol. This showed that stigmasterol was not metabolised during the 72 h of incubation. Also no new metabolites could be identified by MS, confirming that stigmasterol was not metabolised. Weststrate et al. (1999) described a few phytosterol metabolites but this study could not confirm these findings. Conclusions Making use of the newly developed UHPLC-MS method, it was shown that phytosterols are stable under stomach and small intestinal conditions and that they are not metabolised by a bacterial slurry representing the human intestinal microbiota. However, the metabolic activity of the microbiota was negatively influenced by the presence of phytosterols at relevant concentrations as shown by a lower production of SCFA in the presence of phytosterols. These results show an until now unknown, potentially negative effect of phytosterols on the human colonic microbiota, which could potentially affect human health. However, these results need to be confirmed also for other phytosterols, and in vivo. Furthermore, it needs to be investigated if the changes in microbial metabolism have a noticeable effect on human health. In case the results are confirmed a risk-benefit analysis should be performed by the food safety authority (at national or on European level) to determine if the adverse effect of phytosterols on the microbial activity counterbalances the advantages of cholesterol lowering. Gastrointestinal behaviour of phytosterols, K. Struijs, T. Van de Wiele, J. Van Camp; Department of Food Safety and Food Quality, Faculty of Bioscience engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium Laboratory of Microbial Ecology and Technology, Faculty of Bioscience engineering, Ghent University, Coupure Links 653, 9 000 Ghent, Belgium.
