The interaction of gut microbiota with dietary components and its consequences for metabolism

Transcription

1 The interaction of gut microbiota with dietary components and its consequences for metabolism Harry J Flint Microbiology Group, Rowett Institute, University of Aberdeen, UK

2 Prediction of substrate preferences and competition from microbial genomics Relationship between microbiota composition and metabolite outputs Theoretical modelling (influence of ph) Release and transformation of phenolic acids from cereal bran

3 Predictions from bacterial genomes polysaccharide breakdown Among human colonic bacteria, Bacteroides genomes encode the largest numbers of carbohydrate-active enzymes (CAZymes) [El Khaouturi et al Nat Rev Micro (2013)] Does this mean that they make the greatest contribution to degradation of carbohydrate substrates?

4 Glycoside hydrolase genes R. bromii has <10% cf. B. thetaiotaomicron Degradation of dietary resistant starch? B. adolescentis GH5 B. thetaiotaomicron Ent 7L76 GH9 sp unknown SM4/1 C. catus GD/7 R. torques L2-14 R. obeum A2-162 R. obeum SR1/5 C. eutactus ART55/1 B. fibrisolvens 16/4 R. intestinalis XB6B4 R. intestinalis M50/1 E. rectale M104/1 E. rectale DSM17629 E. siraeum V10Sc8a E. siraeum 70/3 F. prausnitzii SL3/3 F. prausnitzii L2-6 GH3 R. bromii L2-63 Rum 18P Number of glycoside hydrolases GH44 GH48 GH10 GH11 GH26 GH43 GH13 other GH (amylases)

5 Glycoside hydrolase genes R. bromii has <10% cf. B. thetaiotaomicron Degradation of dietary resistant starch? B. adolescentis GH5 B. thetaiotaomicron Ent 7L76 GH9 sp unknown SM4/1 C. catus GD/7 R. torques L2-14 R. obeum A2-162 R. obeum SR1/5 C. eutactus ART55/1 B. fibrisolvens 16/4 R. intestinalis XB6B4 R. intestinalis M50/1 E. rectale M104/1 E. rectale DSM17629 E. siraeum V10Sc8a E. siraeum 70/3 F. prausnitzii SL3/3 F. prausnitzii L2-6 GH3 R. bromii L2-63 Rum 18P Number of glycoside hydrolases GH44 GH48 GH10 GH11 GH26 GH43 GH13 other GH (amylases) Bacteroides thetaiotaomicron starch utilization (sus) system (Salyers) susr susa susb susc susd suse susf susg M CM A D G E F C TonB complex B

6 Glycoside hydrolase genes R. bromii has <10% cf. B. thetaiotaomicron Degradation of dietary resistant starch? B. adolescentis GH5 B. thetaiotaomicron Ent 7L76 GH9 sp unknown SM4/1 C. catus GD/7 R. torques L2-14 R. obeum A2-162 R. obeum SR1/5 C. eutactus ART55/1 B. fibrisolvens 16/4 R. intestinalis XB6B4 R. intestinalis M50/1 E. rectale M104/1 E. rectale DSM17629 E. siraeum V10Sc8a E. siraeum 70/3 F. prausnitzii SL3/3 F. prausnitzii L2-6 GH3 R. bromii L2-63 Rum 18P Number of glycoside hydrolases GH44 GH48 GH10 GH11 GH26 GH43 GH13 other GH (amylases) Bacteroides thetaiotaomicron starch utilization (sus) system (Salyers) Ruminococcus bromii has an amylosome (Ze X et al MBio 2015) susr susa susb susc susd suse susf susg M CM A D G E F C TonB complex B

7 Degradation of corn starches by four amylolytic human gut bacteria [Ze X et al 2012 ISME J] Digestible starch (S1) % 80% Resistant starches (S2, S3 (RS2) S4 (RS3)) Ruminococcus bromii (Firmicutes) 60% 40% 20% Bifidobacterium adolescentis (Actinobacteria) Eubacterium rectale (Firmicutes) Grampositive 0% S1 S2 S3 S4 Bacteroides thetaiotaomicron (Bacteroidetes) Gramnegative starches (boiled 10 mins) Bacteroides thetaiotaomicron Ruminococcus bromii susr susa susb susc susd suse susf susg M CM A D G E F C TonB complex B

8 Impact of dietary non-digestible carbohydrate - on dominant species within the faecal microbiota (16S rrna sequencing) :- [+ P <0.001] [Walker A.W. et al (2011). Dominant and diet responsive groups of bacteria within the human colonic microbiota. ISME J 5, ] M Wheat bran Res Starch Weight loss (n =7) M Res Starch Wheat bran Weight loss (n =7) 1 week 3 weeks 3 weeks 3 weeks

9 % residual faecal starch % of universal 16S rrna gene copies Response qpcr of gut analysis microbiota all timepoints to changing ND carbohydrate qpcr - Cluster IV Ruminococcus spp. M NSP RS R WWL P S L Volunteer M RS NSP WL Volunteer incomplete starch fermentation in two people RS diet NSP diet no R. bromii detected R. bromii is a keystone species [Walker AW et al. ISME J 2011 Ze X et al. ISME J 2012] volunteer

10 Conclusions Genome sequence alone is unlikely to tell us which organisms play key roles in substrate degradation For this, we need information from isolated microorganisms and from dietary interventions In the next stage, it may then be possible to identify signature genes and gene products that correlate well with degradative activity

11 Utilization of dietary carbohydrates by the gut microbiota a food chain Insoluble complex carbohydrates ruminococci on faecal fibre (by FISH) Diet Soluble polysaccharides ligosaccharides, sugars Primary degraders = keystone species Polysaccharide utilisers H-utilisers (acetogens, methanogens, SRB); lactate utilisers rganic acids C 2, H 2, CH 4 ligosaccharide/ sugar utilisers (Flint et al Env Micro 2007; Walker et al Env Micro 2008)

12 How do changes in gut microbiota composition impact on metabolite production? Dietary intake, medication, inter-individual variation GUT MICRBITA metabolic pathways community structure GUT ENVIRNMENT cross-feeding METABLIC PRDUCTS

13 The gut microbiota, bacterial metabolites and colorectal cancer [Louis P, Hold GL & Flint HJ 2014 NRM]

14 Impact of bacterially-produced short chain fatty acids on the host Energy sources (acetate, propionate, butyrate) Inhibition of histone de-acetylase (butyrate, propionate) Altered mucosal gene expression, cell differentiation Apoptosis in cancer cells (butyrate) Anti-inflammatory effects (including stimulation of Tregs) Stimulation of host receptors (FFAR2, FFAR3, GPR109..) Influence on gut hormones (eg. GLP-1, PYY..) Influences upon gut transit, gut barrier function Influences on satiety? Peripheral energy supply, lipogenesis (acetate) Protection against colorectal cancer, colitis (butyrate) Prevention of metabolic disease? (butyrate, propionate)

15 Impacts of short chain fatty acid on inflammation and apoptosis acetate, propionate & butyrate differ in: - tissue distribution - recognition - metabolic fate - impact on the host [Louis P, Hold GL & Flint HJ 2014 NRM]

16 Major microbial fermentation pathways methane hydrogen sulphide hexoses & pentoses fucose, rhamnose sulfate H 2 + C 2 PEP DHAP + L-lactaldehyde formate pyruvate oxaloacetate acetate acetyl-coa acetoacetyl-coa C 2 lactate fumarate acetate butyryl-coa ethanol succinate acetyl-coa B2 butyryl-p B1 lactoyl-coa P2 propionyl-coa P1 propionyl-coa propane-1,2-diol P3 propionyl-coa butyrate propionate

17 Microbial propionate & butyrate metabolism (from genomics, metagenomics) Phylogenetic tree (16S rrna sequences) of species for which genomes surveyed Lachnospiraceae Ruminococcaceae Negativicutes Verrucomicrobia Bacteroides 0.05 Roseburia inulinivorans A2-194 Eubacterium rectale A1-86 Roseburia intestinalis M50/1 Ruminococcus gnavus ATCC Coprococcus comes ATCC Ruminococcus torques L2-14 Ruminococcus obeum A2-162 Ruminococcus obeum ATCC Unknown sp. SR1/5 Clostridiales bacterium SS3/4 Clostridium sp. M62/1 Unknown sp. SM4/1 Anaerostipes hadrus SSC/2 Eubacterium hallii L2-7 Coprococcus eutactus L2-50 Coprococcus eutactus ART55/1 Coprococcus catus GD/7 Clostridium lactatifermentans G17 Faecalibacterium prausnitzii L2-6 Faecalibacterium prausnitzii A2-165 Faecalibacterium prausnitzii S3L/3 Phascolarctobacterium succinatutens YIT Veillonella parvula DSM 2008 Megasphaera elsdenii DSM Dialister succinatiphilus YIT Akkermansia muciniphila Muc Bacteroides thetaiotaomicron VPI-5482 Bacteroides vulgatus ATCC 8482 propionate butyrate P3 B2 B2 B2 P3 B1 P3 P3 P3 P3 B2 B2 B2 B2 B2 B1 B1 P2 B2 P2 B2 B2 B2 P1 P1 P2? P1? P1 P1 Metabolic pathways propionate: P1 - succinate P2 - acrylate P3 - propanediol butyrate: B1 - butyrate kinase B2 - acetate:butyryl CoA CoA transferase Reichardt N et al, ISME J (2014)

18 SCFA concentration [mm] Alternative pathways for propionate formation by the human colonic microbiota Utilization of fucose via the propanediol pathway (3) Roseburia inulinivorans A glucose inulin fucose butyrate propionate propanol (Scott KP et al. J Bacteriol 2006) Acrylate route from lactate to propionate (2) Coprococcus catus GD/ Fructose Lactate Fructose + lactate Butyrate Propionate Acetate Lactate

19 % of total clones % propionate Inter-individual variation in Bacteroidetes correlates with % propionate in faecal samples Salonen A et al, ISME J (2014) P1 pathway pyruvate M NSP RS Clone libraries from 1 volunteer [14 male volunteers] mmda 16S rrna C 2 oxaloacetate malate fumarate succinate 10 5 succinyl-coa 0 R-methylmalonyl-CoA S-methylmalonyl-CoA % Bacteroidetes (qpcr) propionyl-coa propionate

20 SCFA Conc. (mm) Impact of ph on SCFA formation by the human colonic microbiota Short chain fatty acids : ph ph 6.5 acetate butyrate total [substrate dietary polysaccharides (mainly starch)] propionate Formate Acetate Propionate I - Butyrate Butyrate ph-controlled I - Valerate continuous Valerate flow Lactate fermentor Succinate Caproate Total Time (hours) Community profile (16S rrna) : E. rectale/roseburia Bacteroides Walker AW et al AEM 2005 Duncan SH et al Env Micro 2009

21 Variation in colonic ph - with anatomical site SmartPill trace [EU SATIN study, U. Aberdeen] Stomach to duodenum ileum to caecum - with time ph Proximal colon lumen ph Main site of carbohydrate fermentation Transverse + distal colon lumen ph Available carbohydrates slowly fermented pressure

22 Major butyrate producers in the colon Phylogenetic tree (16S rrna sequences) of species for which genomes surveyed Lachnospiraceae Ruminococcaceae Negativicutes Verrucomicrobia Bacteroides 0.05 Roseburia inulinivorans A2-194 Eubacterium rectale A1-86 Roseburia intestinalis M50/1 Ruminococcus gnavus ATCC Coprococcus comes ATCC Ruminococcus torques L2-14 Ruminococcus obeum A2-162 Ruminococcus obeum ATCC Unknown sp. SR1/5 Clostridiales bacterium SS3/4 Clostridium sp. M62/1 Unknown sp. SM4/1 Anaerostipes hadrus SSC/2 Eubacterium hallii L2-7 Coprococcus eutactus L2-50 Coprococcus eutactus ART55/1 Coprococcus catus GD/7 Clostridium lactatifermentans G17 Faecalibacterium prausnitzii L2-6 Faecalibacterium prausnitzii A2-165 Faecalibacterium prausnitzii S3L/3 Phascolarctobacterium succinatutens YIT Veillonella parvula DSM 2008 Megasphaera elsdenii DSM Dialister succinatiphilus YIT Akkermansia muciniphila Muc Bacteroides thetaiotaomicron VPI-5482 Bacteroides vulgatus ATCC 8482 Flagellated; major producers of butyrate from dietary ND carbohydrates New species (little known) Lactate-utilizers Decreased in Crohn s disease, anti-inflammatory FIRMICUTES [Barcenilla A et al AEM 2000, Louis P et al J Bact 2004]

23 Major butyrate producers in the colon Phylogenetic tree (16S rrna sequences) of species for which genomes surveyed Lachnospiraceae Ruminococcaceae Negativicutes Verrucomicrobia Bacteroides 0.05 Roseburia inulinivorans A2-194 Eubacterium rectale A1-86 Roseburia intestinalis M50/1 Ruminococcus gnavus ATCC Coprococcus comes ATCC Ruminococcus torques L2-14 Ruminococcus obeum A2-162 Ruminococcus obeum ATCC Unknown sp. SR1/5 Clostridiales bacterium SS3/4 Clostridium sp. M62/1 Unknown sp. SM4/1 Anaerostipes hadrus SSC/2 Eubacterium hallii L2-7 Coprococcus eutactus L2-50 Coprococcus eutactus ART55/1 Coprococcus catus GD/7 Clostridium lactatifermentans G17 Faecalibacterium prausnitzii L2-6 Faecalibacterium prausnitzii A2-165 Faecalibacterium prausnitzii S3L/3 Phascolarctobacterium succinatutens YIT Veillonella parvula DSM 2008 Megasphaera elsdenii DSM Dialister succinatiphilus YIT Akkermansia muciniphila Muc Bacteroides thetaiotaomicron VPI-5482 Bacteroides vulgatus ATCC 8482 Flagellated; major producers of butyrate from dietary ND carbohydrates New species (little known) Lactate-utilizers Decreased in Crohn s disease, anti-inflammatory FIRMICUTES

24 Concentration (mm) Butyrate formation from lactate and acetate by Eubacterium hallii, Anaerostipes hadrus, Anaerostipes caccae 8 [H] 4 lactate 2 H 2 4 pyruvate 2 Fd 2 NADH 2 2 NAD 4 Fd 2 FdH 2 2 FdH 2 4 acetyl-coa 3 CoA 6 [H] + 2 acetyl-coa 4 CoA 4 C Lack of these bacteria may contribute to dysbiosis? Lactate accumulation in stool in colitis patients (Vernia et al 1988) ADP ATP acetyl- P acetate 3 H 2 6 [H] 3 butyryl-coa 2 acetate 5 0 Controls Quiescent colitis Mild colitis Moderate colitis Severe colitis CoA acetyl-coa P i 3 butyrate [Duncan SH et al AEM 2004, Belenguer A et al AEM 2006 Munoz-Tamayo et al FEMS ME 2011]

25 Butyrate formation from lactate and acetate by Eubacterium hallii, Anaerostipes hadrus, Anaerostipes caccae 4 lactate Starch 2 H 2 4 Fd 8 [H] 4 pyruvate 4 CoA Bifidobacterium adolescentis 2 Fd 2 NAD 2 FdH 2 2 FdH 2 4 acetyl-coa 3 CoA + 2 acetyl-coa 4 C 2 metabolite cross-feeding acetate lactate formate 2 NADH 2 6 [H] 3 H 2 6 [H] Eubacterium hallii ATP ADP acetyl- P acetate 3 butyryl-coa 2 acetate butyrate CoA acetyl-coa P i 3 butyrate [Duncan SH et al AEM 2004, Belenguer A et al AEM 2006]

26 Butyrate formation from hexoses in: Roseburia spp., Eubacterium rectale, E. hallii, Anaerostipes spp, Faecalibacterium prausnitzii 8 ADP 8 ATP 4 glucose 8 NAD + 8 NADH Butyryl-CoA: acetate CoA-transferase 8 pyruvate 8 CoA 8 Fd ox 3 ADP 3 ATP 5 butyrate 2 acetate 5 acetate 5 Fd ox 10 NADH 5 Etf 10 NAD + 8 C 2 3 CoA 3 Pi 3 acetyl-p 8 acetyl-coa 5 acetyl-coa 5 butyryl-coa BCD THL 5 CoA 5 acetoacetyl-coa 5 EtfH 2 5 crotonyl-coa 5 H hydroxybutyryl-CoA CR BHBD 5 NADH 5 NAD + 6 H 2 6 Fd ox 13 Fd red 2-7 NAD + 7 NADH 7 Fd ox cytoplasm membrane (Charrier C et al Microbobiology 2006; Louis P & Flint HJ FEMS ML 2009) 14 H + [ -> additional ATP]

27 Functional redundancy functional groups Relative abundance of butyrate-producing Lachnospiraceae in 10 human volunteers:- (based on butyryl-coa:acetate CoA-transferase sequences)* % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% A B C E F H I J K L other A. hadrus coli E. hallii R. intestinalis R. hominis R. inulinivorans R. faecis E. rectale Anaerostipes spp. + Eubacterium hallii Roseburia + E. rectale group *Louis P et al Env Micro 2010

28 Kettle H, Louis P, Duncan SH, Holtrop G, Flint HJ (2015) Modelling the emergent dynamics of communities of human colonic microbiota: response to ph and peptide Environ Microbiol 15: formate H 2 + C 2 B10 CH 4 NSP oligo- starch protein saccharides, sugars B3, B6 B2, B4, B5 B1 B1-B9 PEP B3, B1 pyruvate B1-B9 acetyl CoA B9 acetate B5, B6 B8 succinate B1 lactate B7 Bacterial functional groups :- B1 = Bacteroidetes B2 = Firmicutes (eg. R. bromii) B3 = Firmicutes (eg. E. eligens) B4 = Actinobacteria B5 = Roseburia group B6 = F. prausnitzii B7 = Negativutes B8 = E. hallii, Anaerostipes B9 = acetogens B10 = methanogens butyrate propionate

29 Assigning strain traits/ phenotypes (Helen Kettle) ij H i M ij F b (1 F b ) R R K i K ij (1 R j k J b R K k ik ) ph limitation Resource uptake = μ ij b i /Y ij Max specific growth rate Half-saturation constant Yield Traits (H i, ijm, K ij, Y ij ) for each strain, i, are selected randomly from a uniform distribution over ranges appropriate to each functional group. Each BFG includes 10 strains with varying trait values

30 In our work, empirical data on cultured representatives was used to derive: - Growth rates, saturation constants - Growth yields - Substrate preferences - Fermentation products (including stoichiometry) - Responses to ph Interestingly, it is now possible to gain information on relative growth rates from metagenomic data based on the greater representation of genes close to the replication origin Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples. Korem, T et al Science (2015)]

31 Continuous flow fermentor community (polysaccharide substrates) Metabolic Products [ _ observed; model] Microbiota composition (Walker AW et al AEM 2005) Experiment ph 5.5 ph 6.5 ph 5.5 Model ph 6.5 (Kettle H et al Environ Microbiol 2015)

32 Kettle H, Louis P, Duncan SH, Holtrop G, Flint HJ (2015) Modelling the emergent dynamics of communities of human colonic microbiota: response to ph and peptide Environ Microbiol 15: formate H 2 + C 2 B10 CH 4 NSP oligo- starch protein saccharides, sugars B3, B6 B2, B4, B5 B1 B1-B9 PEP B3, B1 pyruvate B1-B9 acetyl CoA B9 acetate B5, B6 B8 succinate B1 lactate B7 Bacterial functional groups :- B1 = Bacteroidetes B2 = Firmicutes (eg. R. bromii) B3 = Firmicutes (eg. E. eligens) B4 = Actinobacteria B5 = Roseburia group B6 = F. prausnitzii B7 = Negativutes B8 = E. hallii, Anaerostipes B9 = acetogens B10 = methanogens butyrate propionate

33 In silico experiments what are the consequences of if one BFG is missing? Kettle H et al Environ Microbiol (2015)

34 Effects of Atkin s type weight loss diets in obese volunteers [With Alex Johnstone, Gerald Lobley, Rowett HNU] Mean intakes/ day :- Carbohydrate g NSP g Starch g Protein g Fat g Maintenance (M) 400 (52% EI) Moderate carbohydrate (HPMC) 170 (35% EI) Low carbohydrate (HPLC) 23 (4% EI) M (3 days) HPMC (4 weeks) HPLC (4 weeks) M (3 days) HPLC (4 weeks) HPMC (4 weeks) N = 18 male subjects

35 mm [Butyrate] Diet-driven decrease in Roseburia-related bacteria correlates with decreased butyrate in vivo* (faecal samples) 18 obese male volunteers (BMI >30) 30 r = Diets - Low CH Weight Loss (4 wk) Mod. CH Weight Loss (4 wk) High (non WL) CH (3 days) regression log 10 Roseburia/ E. rectale count [*Duncan et al (2007) AEM]

36 mm butyrate Response of SCFA and butyrate-producing bacteria to dietary carbohydrate 120 mm 80 study 1 study 2 Faecal SCFA: isobutyrate isovalerate - two human weight-loss studies in obese males * * valerate butyrate propionate acetate 20 0 M1 HPMC1 LC1 M2 HPMC2 HPLC2 * % Diets: (carbohydrates) 1. Duncan SH et al AEM Russell WR et al Am J Cl Nutr 2011 M HPMC HPLC (normal) (mod) (low) % Roseburia + E. rectale

37 Axis 2 (18% of variance explained) Impact of weight loss diets upon faecal metabolites (PCA plot) Lach-Ros Maintenance Bac M16 Fprau M17 Ros M8 M12 M2 M14 Prop Bu Ac M3M4 M10 M1 NH3 M13 ph High protein, low carbohydrate WL M18 M11 NC M5 M9 Val Lac M6 IsobuIsoval M7 M High protein, moderate carbohydrate WL Axis 1 (29% of variance explained) [Russell WR et al 2011 Am J Clin Nut]

38 Concentration ( g cm -3 ) Impact of diet on plant-derived phenolic acids and microbial metabolites in human volunteers [Russell WR et al AJCN 2011] 4H-3Me-PPA 3,4H-PPA 3H-PPA ferulic acid H H hydrogenation H demethylation H dehydroxylation Faecal concentrations 25 H H H H H P = (means, 8 overweight male volunteers) P < P < ferulic acid metabolite 1 metabolite 2 metabolite 3 diets Maintenance (carbs 400g, NSP fibre 28 g/day) High Protein WL (carbs 170g, NSP fibre 12 g/day) High Protein WL (carbs 23g, NSP fibre 6 g/day)

39 Wheat bran complex insoluble substrate Rich in arabinoxylan Source of ferulic acid - Water absorption faecal bulking, promotion of gut transit - Slowly fermented in the large intestine, yielding SCFA including butyrate and decreasing ph H H - Decrease in carcinogens (eg. secondary bile acids) Lignin R H R R - Source of bioactive phenolic compounds H Feruloyl bridge Arabinoxylan H H H H Bran H

40 Metabolites from pancreatin pre-treated wheat bran SCFA (mm) from substrate fermentation acetate propionate butyrate formate lactate Phenolic acids D D2 0 D1 24 D2 24 D3 24 D4 24 D1 48 D2 48 D3 48 D h 48 h inflow D ferulic acid 3-hydroxy-PPA 4-hydroxy-3- methoxy-ppa 3,4-dihydroxy-PPA D medium [Duncan SH et al Env Microbiol in press] hours

41 % sequences Nine 16S rrna TUs (all Firmicutes) strongly enriched by wheat bran in fermentors (> 5 fold increase in % abundance in at least one person) tu039 tu030 tu029 tu028 tu024 tu017 tu005 tu006 tu007 Firmicutes : 6 Lachnospiraceae 3 Ruminococcoceae 0 2h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h D1 D2 D3 D4 [Duncan SH et al Environ Microbiol, in press]

42 % sequences Nine 16S rrna TUs (all Firmicutes) strongly enriched by wheat bran in fermentors (> 5 fold increase in % abundance in at least one person) Agrees with in vivo data (16S rrna HitChip) [Salonen A et al ISME J 2014] 60 Stimulated by NSP Stimulated by RS tu039 tu030 tu029 tu028 tu024 tu017 tu005 tu006 tu h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h D1 D2 D3 D4 [Duncan SH et al Environ Microbiol, in press]

43 % sequences Wheat bran promotes enrichment within the human colonic microbiota of butyrate-producing bacteria that release ferulic acid Duncan SH et al Environ Microbiol - in press Action of isolated bacteria on wheat bran weight loss (degradation) tu039 tu030 tu029 tu028 tu024 tu017 tu005 tu006 tu007 phenolics ferulic acid 0 2h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h 2h 4h 8h 24h48h D1 D2 D3 D4 tu017-related tu005-related tu006-related

44 Wheat bran degradation ferulic acid and metabolites H H H H hydrogenation demethylation dehydroxylation H H H H (1) (2) (3) (4) H (1) Ferulic acid [4H3Me-CA] (2) 4-hydroxy-3-methoxy phenylpropionic acid [4H3Me-PPA] (3) 3,4-dihydroxy phenylpropionic acid [3,4H-PPA] (4) 3-hydroxy phenylpropionic acid [3H-PPA]

45 Conclusions Many specialist wheat bran degrading bacteria are butyrate-producers and they also release bound ferulic acid during degradation Different bacterial groups are involved in initial release of ferulic acid and in its subsequent transformation Great potential for variation between individuals in gut concentrations of ferulate and ferulate-derived metabolites due to microbiota differences

46 GENERAL CNCLUSINS Genomics and metagenomics need to be complemented by studies on cultured gut isolates Changes in diet composition change the representation of certain diet-responsive species within the microbiota Some are more equal than others (Ze X et al Gut Microbes 2013) (after George rwell). ie. keystone species can play primary roles in degradation of recalcitrant substrates Changes in microbiota composition (due to diet or individual variation) impact on metabolite formation Theoretical modelling can be very helpful in resolving the behaviour of complex microbial communities and their metabolic products

47 Acknowledgements Rowett Institute (U Aberdeen) Alan Walker Sylvia Duncan Petra Louis Karen Scott Xiaolei Ze Nicole Reichardt Alvaro Belenguer Alexandra Johnstone Biomathematics and Statistics Scotland Grietje Holtrop Helen Kettle Support from : Rowett Institute of Nutrition and Health Wellcome Trust Sanger Institute, UK Trevor Lawley Julian Parkhill U Groningen, Netherlands Hermie Harmsen U Helsinki, Finland Anne Salonen Willem de Vos U. Girona, Spain Mireia Lopez-Siles Jesus Garcia-Gill Christian Hansen Cargill