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

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 PGPR.

Steps to reproduce

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’), 806R(5’-GGACTACHVGGGTWTCTAAT- 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 after 48 hours of fermentation was thawed and vortexed for centrifugation. 400 μ L of the supernatant was transferred to a 2 mL centrifuge tube and frozen in liquid nitrogen using a freeze dryer; Add 300 μ L of pre cooled methanol (containing 5 ppm 2-chlorophenylalanine) and vortex for 30 seconds. Place it in a high-throughput tissue grinder at 55 Hz and grind for 60 seconds. Repeat this step once; Put it into the ultrasonic cleaning machine and sonicate for 10 minutes; Freeze in a -20 ℃ freezer for 30 minutes; 12 000 rpm、 Centrifuge at 4 ℃ for 10 minutes, take the supernatant and filter it through a 0.22 μ m membrane. Add the filtered solution to the detection bottle;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.

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Funders

• Shanxi provincial Administration of Traditional Chinese Medicine

  Grant ID: 2024ZYY2C098

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