Low-Fat, High-Fiber Diet Reduces Markers of Inflammation and Dysbiosis and Improves Quality of Life in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: A high-fat diet has been associated with an increased risk of ulcerative colitis (UC). We studied the effects of a low-fat, high-fiber diet (LFD) vs an improved standard American diet (iSAD, included higher quantities of fruits, vegetables, and fiber than a typical SAD). We collected data on quality of life, markers of inflammation, and fecal markers of intestinal dysbiosis in patients with UC. METHODS: We analyzed data from a parallel-group, cross-over study of 17 patients with UC in remission or with mild disease (with a flare within the past 18 mo), from February 25, 2015, through September 11, 2018. Participants were assigned randomly to 2 groups and received a LFD (10% of calories from fat) or an iSAD (35%-40% of calories from fat) for the first 4-week period, followed by a 2-week washout period, and then switched to the other diet for 4 weeks. All diets were catered and delivered to patients' homes, and each participant served as her or his own control. Serum and stool samples were collected at baseline and week 4 of each diet and analyzed for markers of inflammation. We performed 16s ribosomal RNA sequencing and untargeted and targeted metabolomic analyses on stool samples. The primary outcome was quality of life, which was measured by the short inflammatory bowel disease (IBD) questionnaire at baseline and week 4 of the diets. Secondary outcomes included changes in the Short-Form 36 health survey, partial Mayo score, markers of inflammation, microbiome and metabolome analysis, and adherence to the diet. RESULTS: Participants' baseline diets were unhealthier than either study diet. All patients remained in remission throughout the study period. Compared with baseline, the iSAD and LFD each increased quality of life, based on the short IBD questionnaire and Short-Form 36 health survey scores (baseline short IBD questionnaire score, 4.98; iSAD, 5.55; LFD, 5.77; baseline vs iSAD, P = .02; baseline vs LFD, P = .001). Serum amyloid A decreased significantly from 7.99 mg/L at baseline to 4.50 mg/L after LFD (P = .02), but did not decrease significantly compared with iSAD (7.20 mg/L; iSAD vs LFD, P = .07). The serum level of C-reactive protein decreased numerically from 3.23 mg/L at baseline to 2.51 mg/L after LFD (P = .07). The relative abundance of Actinobacteria in fecal samples decreased from 13.69% at baseline to 7.82% after LFD (P = .017), whereas the relative abundance of Bacteroidetes increased from 14.6% at baseline to 24.02% on LFD (P = .015). The relative abundance of Faecalibacterium prausnitzii was higher after 4 weeks on the LFD (7.20%) compared with iSAD (5.37%; P = .04). Fecal levels of acetate (an anti-inflammatory metabolite) increased from a relative abundance of 40.37 at baseline to 42.52 on the iSAD and 53.98 on the LFD (baseline vs LFD, P = .05; iSAD vs LFD, P = .09). The fecal level of tryptophan decreased from a relative abundance of 1.33 at baseline to 1.08 on the iSAD (P = .43), but increased to a relative abundance of 2.27 on the LFD (baseline vs LFD, P = .04; iSAD vs LFD, P = .08); fecal levels of lauric acid decreased after LFD (baseline, 203.4; iSAD, 381.4; LFD, 29.91; baseline vs LFD, P = .04; iSAD vs LFD, P = .02). CONCLUSIONS: In a cross-over study of patients with UC in remission, we found that a catered LFD or iSAD were each well tolerated and increased quality of life. However, the LFD decreased markers of inflammation and reduced intestinal dysbiosis in fecal samples. Dietary interventions therefore might benefit patients with UC in remission. ClinicalTrials.gov no: NCT04147598.

Untargeted metabolomics of the bacterial tongue coating of intra-oral halitosis patients.

Intra-oral halitosis (IOH) refers to an unpleasant odor from the oral cavity that is mainly caused by the tongue coating. Although the tongue coating microbiome is thought to play an essential role in IOH, the exact aetiology of IOH remains unclear. Here we investigated and compared the metabolic profiles of the tongue coating microbiomes of patients with IOH versus healthy control. The metabolic profiles were significantly different in IOH patients than in healthy controls. Healthy controls showed higher selenoamino acid and nicotinamide metabolism; these metabolic pathways are mainly involved in maintaining the oxidation-reduction potential and redox state. A total of 39 putative metabolites were associated with IOH. Remarkably, 3 of the metabolites, branched-chain fatty acids (BCFA), 3-fumaryl pyruvate, and acetyl phosphate, are potential key players in IOH. Interestingly, the predominant metabolite in IOH is BCFAs, which might underlie tongue coat formation. In addition, the key metabolite acetyl phosphate has a clear association with the hydrogen sulfide- (H2S-) producing metabolic pathway and anaerobic fermentation. These novel metabolomic findings provide insights into the formation of the tongue coating and the production of H2S, which causes bad breath.

Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library.

BACKGROUND: Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. RESULTS: Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either pNP-ß-d-galactopyranoside, pNP-ß-d-xylopyranoside, pNP-α-l-arabinopyranoside or pNP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a ß-galactosidase (74%) and (2) a ß-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The ß-galactosidase- and ß-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the ß-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' α-glucosidase activity. CONCLUSION: Using a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.

A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

BACKGROUND: Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. RESULTS: Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. CONCLUSIONS: This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications.

Metasecretome analysis of a lignocellulolytic microbial consortium grown on wheat straw, xylan and xylose.

BACKGROUND: Synergistic action of different enzymes is required to complete the degradation of plant biomass in order to release sugars which are useful for biorefining. However, the use of single strains is often not efficient, as crucial parts of the required enzymatic machinery can be absent. The use of microbial consortia bred on plant biomass is a way to overcome this hurdle. In these, secreted proteins constitute sources of relevant enzyme cocktails. Extensive analyses of the proteins secreted by effective microbial consortia will contribute to a better understanding of the mechanism of lignocellulose degradation. RESULTS: Here, we report an analysis of the proteins secreted by a microbial consortium (metasecretome) that was grown on either wheat straw (RWS), xylose or xylan as the carbon sources. Liquid chromatography-tandem mass spectrometry was used to analyze the proteins in the supernatants. Totals of 768 (RWS), 477 (xylose) and 103 (xylan) proteins were identified and taxonomically and functionally classified. In RWS, the proteins were mostly affiliated with Sphingobacterium-like consortium members (~50 %). Specific abundant protein clusters were predicted to be involved in polysaccharide transport and/or sensing (TonB-dependent receptors). In addition, proteins predicted to degrade plant biomass, i.e. endo-1,4-beta-xylanases, alpha-l-arabinofuranosidases and alpha-l-fucosidases, were prominent. In the xylose-driven consortium, most secreted proteins were affiliated with those from Enterobacteriales (mostly Klebsiella species), whereas in the xylan-driven one, they were related to Flavobacterium-like ones. Notably, the metasecretomes of the consortia growing on xylose and xylan contained proteins involved in diverse metabolic functions (e.g. membrane proteins, isomerases, dehydrogenases and oxidoreductases). CONCLUSIONS: An analysis of the metasecretomes of microbial consortia originating from the same source consortium and subsequently bred on three different carbon sources indicated that the major active microorganisms in the three final consortia differed. Importantly, diverse glycosyl hydrolases, predicted to be involved in (hemi)cellulose degradation (e.g. of CAZy families GH3, GH10, GH43, GH51, GH67 and GH95), were identified in the RWS metasecretome. Based on these results, we catalogued the RWS consortium as a true microbial enzyme factory that constitute an excellent source for the production of an efficient enzyme cocktail for the pretreatment of plant biomass.