Fiber-enriched Centella asiatica longitudinally suppresses proteolytic fermentation and reprograms microbe–metabolite networks
Description
Human microbiome intervention studies often focus on taxonomic shifts, whereas longitudinal changes in functional metabolic outputs remain less defined. In this randomized trial, dietary fiber enrichment of a traditional Centella asiatica beverage selectively suppressed branched short-chain fatty acids (BSCFAs), markers of proteolytic fermentation, in healthy adults. Integrating microbiome profiling, targeted metabolomics, and microbe–metabolite network analysis, we show that fiber availability acts as a dominant driver of metabolic reprogramming, favoring saccharolytic over proteolytic pathways without major compositional restructuring. These findings provide longitudinal evidence that targeted fiber supplementation modulates BSCFAs as functional indicators of microbial metabolic plasticity in urban dietary settings.Human microbiome intervention studies mostly emphasize on taxonomic and computational changes, while functional microbial metabolic outputs and their longitudinal dynamics remain less well characterized. Our longitudinal randomized trial demonstrates that dietary fiber enrichment of a traditional Centella asiatica beverage selectively suppresses branched short-chain fatty acids (BSCFAs), key markers of proteolytic microbial fermentation, in healthy adults. By integrating gut microbiome profiling with targeted metabolomics, and microbe–metabolite network analysis, we show that fiber availability can acts as a dominant driver of microbial metabolic reprogramming, favoring saccharolytic over proteolytic pathways even in the absence of overt clinical changes. This study provides high-resolution longitudinal evidence that dietary fiber supplementation modulates BSCFAs as functional readouts of microbial metabolic reprogramming in healthy adults, offering mechanistic insight into how targeted fiber interventions influence the metabolic profile of the gut microbiota in urbanized dietary contexts.
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Steps to reproduce
1. Fecal SCFAs (GC-TOFMS) • Extraction: Use acidified solvent extraction on fecal samples. • Internal Standard (IS): Spike with stable isotope-labeled IS. • QC: Prepare a pool of all samples; run periodically to monitor stability/reproducibility. • Analysis: Pegasus BT GC-TOFMS (LECO) with an FFAP column. • Software: ChromaTOF for data acquisition and processing. 2. Total Plasma Fatty Acids (GC-TOFMS) • Derivatization: Perform esterification/methylation using BF₃–methanol. • IS: Use nonadecanoic acid (C19:0). • Extraction: Hexane extraction following derivatization. • Analysis: Pegasus BT system with a ZB-FAME column. • Quantification: 10-point external calibration curve (0.05–100 ng/µL). • Validation: Include SRM plasma and pooled plasma QCs. 3. Plasma Cholesteryl Esters (CEs) (UPLC-MS) • Initial Extraction: 20 µL plasma + MTBE:methanol (5:1, v/v) with 50 µM cholesterol-d7 IS. • Processing: - Manual vortex (1 min) → Multi-tube vortex (2,000 rpm, 15 min). - Sonicate (35°C, 15 min) → Centrifuge (12,000 rpm, 15 min, 4°C). - Evaporate supernatant (70°C, 30–40 min). • Reconstitution: Acetonitrile:Isopropanol (60:40, v/v); sonicate at 35°C. • Chromatography: o Column: RP (2.1 x 100 mm, 1.7 µm) at 55°C. o Mobile Phase: A (ACN/H₂O + AmFm/FA) and B (IPA/ACN + AmFm/FA). o Gradient: 60% A → 1% A (15 min) → 99% A (re-equilibration); Total: 18 min. o Flow: 0.3 mL/min; Injection: 5 µL. • Stability Tip: Keep autosampler at 10–15°C to prevent lipid precipitation. 4. Plasma Uremic Toxins (UPLC-TQ-MS) • Extraction: Organic solvent extraction with isotope-labeled IS. • Dilution: Perform additional dilutions to ensure metabolites stay within the linear range of the detector. • Analysis: Waters Xevo TQ-S (Triple Quadrupole). • Separation: ACQUITY BEH HILIC column. • Detection: Electrospray ionization (ESI) in both positive and negative modes.
Institutions
- Mahidol UniversityBangkok, Bangkok