Nami Baskota | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Nami Baskota. This seminar is open to the general public to attend.
Thesis Topic: Determination of dietary fibre fermentation by the human gut microbibiota using a simple in vitro batch model

MSc with Dr. Jens Walter

Seminar Abstract:

Dietary fibre (DF), which is not digested by mammalian enzymes, constitutes a critical substrate for bacterial fermentation in the gastrointestinal tract, resulting in short-chain fatty acids (SCFAs) production. However, the ability of the human gut microbiome to ferment specific fibre structures is highly individualized, and little is known about how differences in fibre fermentation in individuals influence the production of SCFAs. The primary objective of my research was to optimize a batch in vitro fermentation model and apply the model in human trials to characterize the importance of structural differences of DF for the production of SCFAs by the human gut microbiota.

For optimization of a batch IVFF model, two fecal inoculum concentrations 5% and 2% w/v and three nutrient media, Medium 1, Medium 2 and Medium 3 were used. Medium 1 was the base medium, while Medium 2 and 3 were base medium with concentration of peptone, yeast extract and Tween 80 reduced to 20% and 10% of their concentration in the base medium. Fecal samples from three healthy individuals were used for in vitro fermentation with Resistant starch type 4 (RS4). Fermented sample was measured for SCFAs and branched chain fatty acids (BCFAs) via Gas Chromatography (GC) and the growth of Escherichia coli (E. coli) during fermentation was quantified by plating. The concentration of fecal inoculum had no effect on the absolute amount of SCFAs and BCFAs produced and growth of E. coli during the fermentation. The concentration of total SCFA relative to control increased by 1.77 times (p< 0.05) while concentration of total BCFA relative to control decreased by 0.04 times (p < 0.05) in Medium 3 as compared to Medium 1. In addition, there was a 2 log (p < 0.05) and 4 log (p < 0.05) reduction in the growth of E. coli in Medium 2 and Medium 3 as compared to its growth in Medium 1. The optimized batch IVFF model was repeatable as the average coefficient of variation for acetate, propionate, and butyrate were less than 12% for technical replicates when pectin (PE) was fermented. These findings provide evidence that the performance of the in vitro model could be optimized by reducing the concentration of peptone and yeast extract in the medium.

The second goal of the thesis was to determine the ability of the batch IVFF model to assess the inter-individuality of gut microbial community to ferment chemically distinct fibres to SCFAs. Isolated DFs such as arabinoxylan, pectin, RS4, acacia gum and microcrystalline cellulose were subjected to in vitro upper gastrointestinal digestion and human fecal fermentation and measured over 14 hours for SCFAs. Significant differences were found between SCFAs produced by the different fibres with a high degree of variability between the subjects. The third goal was to determine the impact of a six-week high daily dose of arabinoxylan supplementation on the capacity of the gut microbiota of overweight individuals to ferment the fibre into SCFAs. Healthy overweight subjects (n=31) consumed AX or MCC (control) either 25g/d or 35g/d, women and men respectively, for six weeks. Fecal samples were collected at baseline, week 1 and week 6. SCFAs and BCFAs were quantified directly in the fecal samples and through in vitro fermentation. There was a significant increase in molar proportion of fecal propionate at week 6 as compared to baseline (p = 0.01) but the in vitro measurements did not show any shifts in SCFA production, reflecting the failure of the batch IVFF model to confirm with the in vivo findings.

Together findings in this dissertation indicate that the batch IVFF model is able to study the fermentation of chemically distinct fibres by fecal microbiota however the model seems to have very limited suitability to study more subtle differences in fermentation, such as those induced through feeding of DF.