Abstract
Increased intestinal permeability is linked to low-grade systemic inflammation associated with chronic diseases. Undigested dietary proteins reach the colon, where they are fermented by components of the gut microbiota to produce metabolites shown to increase intestinal permeability in vitro. As evidence for sex differences in the microbiota grows, we hypothesised that the effects of the microbial fermentation of protein would also be sex-dependent. Thus, our objective was to determine whether there were sexual dimorphisms in microbial composition and metabolic output following the fermentation of different proteins using in vitro human gut model systems. Faeces from healthy male (n = 5) and female (n = 5) donors were used to inoculate gut fermentation systems supplemented with non-hydrolysed proteins (0.9 g) derived from whey, fish, milk, soya, mycoprotein, egg or pea. At 0, 8, 24 and 48 h, the microbiota composition was quantified using fluorescence in situ hybridisation coupled with flow cytometry, while bacterial-derived metabolite production was assessed via gas chromatography/mass spectroscopy and an ELISA. Increased protein availability resulted in significant increases in proteolytic Bacteroides spp. (p < 0.01) and Clostridium coccoides (p < 0.01) and significant increases in the production of potentially detrimental metabolites including phenol (p < 0.01), p-cresol (p < 0.01), indole (p = 0.018) and ammonia (p < 0.01), all of which were highly dependent on protein type. Furthermore, we showed higher abundances of Clostridium cluster IX (p = 0.03) and concentrations of p-cresol (p = 0.025) at 24 h in males, while females produced more ammonia (p = 0.02) irrespective of the protein source. The fermentation of mycoprotein resulted in significantly higher abundances of Clostridium cluster IX in males at 8 and 24 h compared to females (p < 0.01). There were also significant interactions between sex, protein source, bacterial populations and bacterial-derived metabolic-end-product concentrations. Our study provides new evidence that the effects of the microbial fermentation of dietary proteins in vitro are highly dependent on the source of the protein and the sex of the donor. Consequently, we suggest that different proteins are likely to have differential impacts on intestinal barrier function in vivo, and these effects may be different in males and females. If corroborated in human studies, our results would have important implications for dietary recommendations to limit chronic diseases.
Author Contributions
M.C.L., M.D.R. and B.A.G. designed the project and won the grant application. J.G. helped with the grant application and procured reagents used in the experiments. D.J. conducted the in-vitro experiments. D.J. conducted microbiota and short-chain fatty acid analysis. G.E.W. supported with the in-vitro experiments and provided insight on the interpretation of microbiota and metabolite analyses. J.S.E. conducted the phenolic compound analysis using SPME-GCMS and wrote the methods section for this specific measurement. D.J. and M.C.L. wrote the main manuscript text and prepared all figures. All authors have read and agreed to the published version of the manuscript.
Funding
This work was primarily funded by the BBSRC under Grant BB/T008776/1 with additional funding from Food and Feed Innovations.
Institutional Review Board Statement
Not Applicable.
Informed Consent Statement
Informed consent was given by all individuals who provided samples.
Data Availability Statement
The data that support the finding of this study are openly available in University of Reading Data Archive at https://doi.org/10.17864/1947.000504.
Conflicts of Interest
The authors declare that this study received funding from Food and Feed Innovations. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
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