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Fermentation And behaviour of carbohydrates in the colon

Periodic Reporting for period 1 - FABCARB (Fermentation And behaviour of carbohydrates in the colon)

Reporting period: 2017-04-01 to 2019-03-31

Problem being addressed:
IBS is a complex syndrome without a known cause or clear diagnostic markers, although it has often been linked to issues with diet or digestive transit time. Recently, research has focussed in particular on the role of the microbiome, which is thought to be altered in many IBS patients, leading to altered carbohydrate ultilisation. As a result, many clinical guidelines suggest IBS sufferers avoid the consumption of a common form of fermentable carbohydrate, resistant starch (RS). There is not, however, a mechanistic basis to understand the difference in RS fermentation between healthy individuals and IBS suffers. This project aimed to address this through identifying mechanistic differences in RS fermentation between healthy and IBS patient microbiota.

Why is it important for society:
IBS is a major societal problem, as one of the most widely diagnosed disorders in Europe. Although not fatal, the associated deterioration in quality of life and co-morbidities pose major challenges for health care systems. There is currently no known cure for IBS, and the economic and healthcare costs are highly significant, as well as the impact on quality of life for individual sufferers. Current interventions for IBS are primarily diet based, for example the commonly used FODMAP diet, which aims to restrict fermentable carbohydrate intake. This diet, although successful in helping many IBS sufferers, is highly restrictive and the long-term health effects of removing fermentable carbohydrates from the diet are unclear. Therefore, there is a pressing societal need for improved dietary interventions in IBS, which can be informed by a better mechanistic understanding of the fermentation of different carbohydrates by the IBS microbiota.

Overall objectives:

The overall objectives of this project are:
1. Selecting a range of RS from the same botanical origin, but with different structural processing resulting in different mechanisms of resistance to digestion
2. Obtaining healthy and IBS patient faecal inoculum, including ethical approval for the study, and using these samples to seed ex vivo model colon systems containing contrasting RS substrates and analysing the kinetics of gas production during fermentation of the RS substrates.
3. Carrying out metabolomic and next generation 16S sequencing analysis on samples of media taken from fermentation vessels during the time course of fermentation of different RS substrates with healthy or IBS faecal inoculum to identify differences in microbial community composition and carbohydrate metabolism.
Objective 1. Selecting a range of RS from the same botanical origin, but with different structural processing resulting in different mechanisms of resistance to digestion

Three substrates were selected based on chickpea, as this choice of material allowed contrasting structural features to be generated from starting material of the same botanical origin. The structure of these substrates were characterised in detail using differential scanning calorimetry and 13C CP/MAS NMR, revealing differences in starch crystallinity between substrates. Metabolomic analysis identified maltose and glucose produced as a result of fermentation.

2. Obtaining healthy and IBS patient faecal inoculum, including ethical approval for the study, and using these samples to seed ex vivo model colon systems containing contrasting RS substrates and analysing the kinetics of gas production during fermentation of the RS substrates.

Ethical approval for the study was successfully sought through the UEA REC and faecal samples were collected via the Norwich Biorepository service, in collaboration with clinicians at the Norfolk and Norwich University Hospital. 13 healthy and 8 IBS samples were collected and inoculated into model.
There were clear statistical differences in gas production rate driven by the different substrate structures. Interestingly, no significant differences were observed between healthy and IBS patient fermentation's for any of the substrates, suggesting within the limits of this study, that for RS there is not a microbiota driven difference in gas production between IBS patients and healthy individuals.

3. Carrying out metabolomic and next generation 16S sequencing analysis on samples of media taken from fermentation vessels during the time course of fermentation of different RS substrates with healthy or IBS faecal inoculum to identify differences in microbial community composition and carbohydrate metabolism.

Metabolite analysis has been carried out using 1H NMR spectroscopy. In total 24 metabolites were identified and quantified in our metabolite screen. Clear differences could be observed between the substrates, in particular in first steps of starch hydrolysis with the release of maltose and glucose in the early stages of fermentation.

Overview of the results and their exploitation and dissemination

The main outcome of this research is the finding that there is no significant difference in gas production during fermentation between microbiota from healthy and IBS patient individuals. This is a striking and novel finding, which supports recent in vivo findings by other workers also indicating that microbial gas production may not explain the pain and bloating symptoms experienced in IBS. These results are currently in preparation for a publication, and have also been used to power a future study in collaboration with King’s College London and University of Nottingham where we are combining in vitro fermentation experiments with human studies using MRI imaging to identify the cause of bloating in IBS.
The results of this project are providing a unique and surprising insight into the microbiota of patients with IBS. It has regularly been inferred from previous data around bloating and abdominal pain in IBS, as well as the apparently beneficial effects of removing fermentable carbohydrates from the diets of IBS patients, that microbial gas production is implicated in IBS. This study, has, however, provided evidence that in an isolated, ex vivo system there is no significant difference between healthy and IBS patient microbiota in terms of the kinetics or volume of gas production. This suggests that gas production alone will not explain how differences in microbiota composition in IBS can influence symptoms in IBS. An important next step in analysing the outcomes of this project will be to combine the metabolomics data with 16S community composition data (these data sets are currently being analysed). This will allow me to integrate the metabolite profiles and bacterial communities during starch fermentation, to identify if there are clear differences in metabolites other than gas which may play a role in the development of symptoms following carbohydrate consumption in IBS patients.

The MSCA Fellowship has allowed me to establish a research group within the Quadram Institute, and I am now a tenure track faculty member following the conclusion of my Fellowship. Through this I will be able to continue the research I have begun as part of my Fellowship into the role of fermentable carbohydrates in IBS, and into the mechanisms of carbohydrate fermentation more broadly. As a result of the outcomes of the FABCARB Fellowship I have been able to design two further studies with clinical partners to explore the results in a clinical setting and add a translational aspect to the research.
Gas kinetics measurements being taken, and outcomes of gas kinetics results for healthy and IBS