Analysis of intestinal flora composition and non-targeted metabolomics of PRTP in vitro fermentation

Published: 24 November 2025| Version 1 | DOI: 10.17632/kzyzgdbzct.1
Contributor:
xi peng

Description

The composition of gut microbiota was explored through 16s rRNA sequencing, and non-targeted metabolomics was employed to detect the types of metabolites in the in vitro fermentation study of PRTP.

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Following 48 h of fermentation, genomic DNA was isolated from the fermented sediment utilizing the MagBeams FastDNA Kit for Soil (MP Biomedicines, CA, USA). PCR amplification of the V3-V4 hypervariable regions within the 16S rRNA gene was performed with locus-specific primers of 338F (5 '- barcode+ACTCCTACGGGAGGCAGCA-3') and 806R (5 '- GACTACHVGGGTWTCTAAT-3'). Subsequent to PCR amplification, sequencing reactions were carried out with the NovaSeq 6000 SP Reagent Kit on an Illumina NovaSeq sequencing platform. Vsearch was utilized to perform manipulating taxonomic units (OUTs) clustering with a similarity level of 97%. The supernatant collected at 48 h of fermentation (200 μL) was thoroughly combined with 1 mL of pre-chilled methanol: acetonitrile (1 : 1, v/v). The sample was filtered through a 0.22 μm microporous membrane prior to injection into an UHPLC hyphenated with a high-resolution mass spectrometer (Thermo Fisher Scientific, USA). The analytical column employed was an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm), with a flow rate of 0.4 mL/min, column temperature of 40 ℃, automatic sampler at 8 ℃, and injection volume of 2 μL. In positive and negative modes, mobile phase A was 0.1% formic acid water, and mobile phase B was acetonitrile (containing 0.1% formic acid). Elution was performed using the following gradient program: 0 min, 5% mobile phase B; 1 min, 5% mobile phase B; 4.7 min, 95% mobile phase B; 6 min, 95% mobile phase B; 6.1 min, 5% mobile phase B; 8.5 min, 5% mobile phase B. MS-DIAL (version 4.9.221218) was employed for peak extraction, alignment, filtration, metabolite characterization, and other operations. Metabolites were identified based on the PerSonalbio Next Generation Metabolomics Database (PSNGM) database, which included self-built standard libraries, mzCloud libraries (https://www.mzcloud.org/), LIPID MAPS (https://www.lipidmaps.org/), HMDB (https://hmdb.ca/), MoNA (https://mona.fiehnlab.ucdavis.edu/), NIST2020_SMS and AI predicted MSMS graph library.

Categories

Metabolism, Gut Microbiome

Funders

  • Shanxi provincial Administration of Traditional Chinese Medicine
    Grant ID: NO. 2023ZYYC059

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