Determination of dietary fibre fermentation by the human gut microbiota using an in vitro batch model

• Author / Creator

  Baskota, Nami

• 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 DF structures is highly individualized, and little is known about how inter-individual differences in DF fermentation influence the health effects of DF. The primary objective of my research was to optimize a batch in vitro fermentation model and apply the model to human trials to characterize the importance of structural differences of DFs on the production of SCFAs by the human gut microbiota.
  For optimization of a batch in vitro model, two fecal inoculum concentrations (5% and 2% w/v) and three growth media (Medium 1, Medium 2 and Medium 3) were tested. Medium 1 was a medium similar to what has been used in the literature, while Medium 2 and 3 used the same recipe but concentrations of peptone, yeast extract and Tween 80 were reduced to 20% and 10%, respectively. Fecal samples from three healthy individuals were used for in vitro fermentation (14 hours) with resistant starch type 4 (RS4) as a carbon source or no added carbohydrate (control). SCFAs and branched-chain fatty acids (BCFAs) were determined using 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 SCFAs relative to control increased by 2.16-fold (p  0.05) while the concentration of total BCFAs relative to control decreased by 16-fold (p  0.05) in Medium 3 as compared to Medium 1. In addition, there was a 4.54-log (p  0.05) reduction in the growth of E. coli in Medium 3 as compared to its growth in Medium 1. These findings provide evidence that the growth medium with the concentration of peptone and yeast extract reduced to 10% of their concentration commonly used in the literature resulted in an improved ability to detect SCFAs production from DF fermentation, reduced production of total BCFAs, and in an almost complete prevention in the growth of E. coli.
  The second goal of the thesis was to determine the ability of the batch IVFF model to assess the capacity of fecal microbial communities of different individuals to ferment chemically distinct DFs to SCFAs. The isolated DFs arabinoxylan (AX), acacia gum, pectin, RS4, and microcrystalline cellulose (MCC) were subjected to an in vitro digestion mimicking conditions in the small intestine and in vitro fecal fermentations as described above. Significant differences were found between SCFAs produced by the different DFs with a high degree of variability between the subjects confirming that significant inter-individual differences occur. The third goal was to determine the impact of a six-week supplementation of a high daily dose of AX on the capacity of the gut microbiota of overweight individuals to ferment the DF into SCFAs. Healthy overweight subjects (n=31) consumed AX or MCC (control), either 25g/d or 35g/d for women and men respectively, for six weeks. Fecal samples were collected at baseline, week 1, and week 6. SCFAs were quantified directly in the fecal samples and using the in vitro fermentation method. There was a significant increase in the molar proportion of propionate in the fecal sample at week 6 as compared to baseline (p  0.05) in the AX arm. An effect of AX was not detected using in vitro fermentations. Thus, the batch IVFF approach was less sensitive to study the response of gut microbiota to AX supplementation, especially in terms of propionate production.
  Together, the findings in this dissertation indicate that the batch IVFF model was successfully optimized, which could be used to study the fermentation of chemically distinct DFs by fecal microbiota. The batch IVFF model developed here was already successfully used in one published study (Jin et al., 2019) and is used in an ongoing study in the Walter lab, demonstrating that it provides a platform for future nutrition studies to assess individualized host responses to DF fermentation to eventually personalize the use of DF based on individual microbiome function.

• Subjects / Keywords

  • short-chain fatty acids
  • gut microbiota
  • in vitro batch model
  • Dietary Fibre
  • fermentation

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-75qg-va55

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.