Wheat Bran Promotes Enrichment Within the Human Colonic Microbiota of Butyrate-Producing Bacteria That Release Ferulic Acid
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Aberdeen University Research Archive Environmental Microbiology (2016) 18(7), 2214–2225 doi:10.1111/1462-2920.13158 doi:10.1111/1462-2920.13158 Wheat bran promotes enrichment within the human colonic microbiota of butyrate-producing bacteria that release ferulic acid Sylvia H. Duncan,1 Wendy R. Russell,1 phenylpropionic acid derivatives via hydrogenation, Andrea Quartieri,2 Maddalena Rossi,2 demethylation and dehydroxylation to give metabo- Julian Parkhill,3 Alan W. Walker1,3*† and lites that are detected in human faecal samples. Pure Harry J. Flint1† culture work using bacterial isolates related to the 1Rowett Institute of Nutrition and Health, University of enriched OTUs, including several butyrate-producers, Aberdeen, Aberdeen, UK. demonstrated that the strains caused substrate 2Department of Life Sciences, University of Modena and weight loss and released ferulic acid, but with limited Reggio Emilia, Modena, Italy. further conversion. We conclude that breakdown of 3Pathogen Genomics Group, Wellcome Trust Sanger wheat bran involves specialist primary degraders Institute, Hinxton, Cambridgeshire, UK. while the conversion of released ferulic acid is likely to involve a multi-species pathway. Summary Introduction Cereal fibres such as wheat bran are considered to offer human health benefits via their impact on the Non-digestible fibre such as cereal bran is considered to intestinal microbiota. We show here by 16S rRNA be an important component of a healthy human diet gene-based community analysis that providing (Nilsson et al., 2008; Smith and Tucker, 2011; Flint et al., amylase-pretreated wheat bran as the sole added 2012). Fibre can affect health through multiple mecha- energy source to human intestinal microbial commu- nisms, which include its influence on gut transit, prebiotic nities in anaerobic fermentors leads to the selective effects, its fermentation in the large intestine to yield and progressive enrichment of a small number of short-chain fatty acids (SCFA), and its contribution to the bacterial species. In particular, OTUs corresponding host metabolome of potentially health-protective com- to uncultured Lachnospiraceae (Firmicutes) related to pounds of plant origin (Lampe et al., 1992; Gill and Eubacterium xylanophilum and Butyrivibrio spp. were Rowland, 2002; Dhingra et al., 2012). Most of these strongly enriched (by five to 160 fold) over 48 h in four effects depend on the activities of the resident microbiota independent experiments performed with different within the large intestine, which has the capacity to faecal inocula, while nine other Firmicutes OTUs degrade fibre that cannot be digested by the host’s diges- showed > 5-fold enrichment in at least one experi- tive enzymes. Feeding of wheat bran has been shown to ment. Ferulic acid was released from the wheat bran decrease chemically induced tumours in rodents, and this during degradation but was rapidly converted to effect has been ascribed largely, but not exclusively, to the stimulation of butyrate formation via wheat bran fermen- Received 2 September, 2015; revised 9 November, 2015; accepted tation in the large intestine (McIntyre et al., 1993; Zoran 27 November, 2015. *For correspondence. E-mail alan.walker@ et al., 1997; Compher et al., 1999; Damen et al., 2011; abdn.ac.uk; Tel. +44 (0)1224 438739; Fax +44 (0)1224 438699. †These authors contributed equally to this work. Author contributions: Mikkelsen et al., 2011; Louis et al., 2014). HJF, WRR, AWW and SHD devised the study. AQ and SHD per- Microbial degradation of cereal bran also leads to the formed the experiments. HJF, AWW, WRR and SHD analysed the release of phenolic compounds that are detected in faecal data and prepared the figures and tables. JP and MR provided critical resources and support. HJF, AWW, SHD and WRR wrote the paper. samples (Russell et al., 2011), e.g. compounds such as All authors read and approved the final manuscript. Originality signifi- ferulic acid, which are integral components within plant cance statement: In this work we identify human colonic bacteria that cell wall structures (Bunzel et al., 2001; Manach et al., are capable of degrading wheat bran. Key wheat bran degraders are revealed to be predominantly derived from the Firmicutes phylum, 2004). These compounds can be taken up into the circu- and include butyrate producers. The promotion of particular butyrate- lation and therefore have the potential to influence producing bacteria within a diverse microbial community, together systemic health in addition to gut health. Phenolic com- with the release of bioactive phenolic compounds during wheat bran degradation, can potentially explain many of the health benefits that pounds can act through a variety of mechanisms, includ- are attributed to cereal fibres. ing as antioxidants, upregulators of host redox systems, V©C 2015 The Authors. Environmental Microbiology Microbiology published published by by Society Society for for Applied Applied Microbiology Microbiology and and John John Wiley Wiley & & Sons Sons Ltd. Ltd. This is an open access article under the terms of the Creative Commons Commons Attribution Attribution License, License, which which permits permits use, use, distribution distribution and and reproduction in any medium, medium, provided provided the the original original work work is is properly properly cited. cited. 2 S. H. Duncan et al. Wheat bran degradation by the human colonic microbiota 2215 as anti-inflammatory agents and as promoters of sample provided by a healthy donor; samples were taken apoptosis in cancer cells (Stevenson and Hurst, 2007; after 2, 4, 8, 24 and 48 h incubation and processed to Islam et al., 2008). These diverse mechanisms are yield S (solid substrate) and L (liquid) fractions (see thought to underlie the epidemiological and experimental Experimental procedures section). Four experiments evidence of protection against cardiovascular disease were conducted, each with a different adult faecal donor. and cancer from increased fibre consumption (World In all cases an increase in colonization of the substrate by Cancer Research Fund/American Institute of Cancer bacteria, especially by Lachnospiraceae, during incuba- Research report, 2007; Louis et al., 2014). While some tion was clearly evident from fluorescent in situ hybridiza- phenolic compounds are derived from the fermentation of tion (FISH) analysis (Fig. S1). The DNA extracted from aromatic amino acids, plant fibre appears to be the major each sample was amplified with barcoded primers target- source of phenolic compounds that are considered to ing the 16S rRNA gene, and the resulting amplicons benefit health (Jenner et al., 2005; Russell et al., 2011; (V3–V5 regions) were then sequenced using 454 2013). pyrosequencing. Detailed analysis at the operational There is surprisingly little understanding of the microbial taxonomic unit (OTU) level (97% cut-off) revealed a total ecology of fibre breakdown in the human colon, particu- of 406 OTUs, of which 14 accounted individually for > 2% larly for complex, insoluble, forms of fibre such as cereal and 23 for > 1% of total sequences. As differences bran that consist primarily of plant cell walls (Faulds, between S and L fractions were shown to be insignificant 2010). Previous studies showed that different bacterial using METASTATS, the summed (S + L) data for each time species were promoted in the faecal microbiota of human point are considered here. volunteers fed controlled diets enriched with wheat bran Overall bacterial community diversity, as assessed by compared with resistant starch (Walker et al., 2011; calculating observed OTU richness, Chao estimates of Salonen et al., 2014). This agrees with earlier work total richness, and the Shannon and Simpson diversity showing that different bacterial 16S rRNA gene indices, was not significantly different between fermentor sequences became enriched in incubations with insoluble systems at each sampling time point, with mean Shannon wheat bran, resistant starch or porcine mucin in an in vitro indices of around 3 at 24 and 48 h (Fig. 1A). There was, fermentor system, indicating a high degree of substrate however, marked donor-specific variation in microbiota specificity among the species colonizing these substrates composition between fermentor experiments (Analysis of (Leitch et al., 2007). Release of phenolic acids from wheat molecular variance (AMOVA), P = 0.006). Incubation with bran by human gut microbiota has been demonstrated in wheat bran also had a significant impact on the temporal vitro using batch cultures (Nordlund et al., 2012). community structure within the fermentors (AMOVA, Our objective here was to analyse the colonization of P = 0.041), and the impacts of both inter-donor variation wheat bran by human colonic microbiota. For this we used and incubation with wheat bran on microbiota composition the in vitro fermentor system described by Leitch and were visualized using principal coordinates analysis colleagues (2007), in which the insoluble substrate is (PCoA) (Fig. 1B). More specifically, nine OTUs (all incubated under conditions of controlled pH and a con- Firmicutes) were identified as becoming strongly enriched tinuous input of sterile anaerobic medium. We report the (defined as >5-fold increase in proportional abundance) sequence of changes in microbiota composition with time among amplified 16S rRNA gene sequences at the 24 or during colonization of wheat bran using high-throughput 48 h time points relative to the 4 h time point (Fig. 2A, 16S rRNA gene sequencing and identify for the first time Fig. S2). Across the four experiments these nine OTUs, the major species likely to be responsible for wheat bran taken together, accounted for 1.5–5.5% of all sequences degradation in the human colon. At the same time, we at 2 h and 4 h (means: 1.9% and 3.2%, respectively), were able to follow the release and transformation of 4–16% (mean: 8.3%) at 8 h, 27–36% (mean: 32.6%) at wheat bran-derived phenolic metabolites by the associ- 24 h, and 27–52% (mean: 40%) at 48 h. In contrast, 11 of ated microbial community.