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Page 4 of 15 Lai et al. Microbiome Res Rep 2024;3:21 https://dx.doi.org/10.20517/mrr.2023.76

10 min, and 1 mL of the supernatant was filtered through a 0.22 μm filter membrane. OAs separation was
done on an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad) at a column temperature of 35 °C
using H SO (0.004 M) as the mobile phase at a wavelength of 215 nm. The injection volume was 20 μL and
4
2
the flow rate was adjusted to 0.6 mL/min.

Non-target metabolomics analysis
The fermented mustards (100 ± 5 mg) were mixed with 400 μL extraction solution (80% methanol-water
solution, v/v) containing 0.02 mg/mL L-2-chlorophenylalanine (internal standard). Samples were separately
ground using Wonbio-96c frozen tissue grinder (Shanghai Wanbo Biotechnology Co., LTD.) for 6 min
(-10 °C, 50 Hz), and then extracted by low-temperature ultrasound for 30 min (5 °C, 40 kHz). Subsequently,
the sample was centrifuged for 15 min (4 °C, 13,000 g) after being left at -20 °C for 30 min, and the
supernatant was transferred to the injection vial for ultra-high performance liquid chromatography-tandem
mass spectrometry (UPLC-MS/MS) analysis. In addition, the mixture of each supernatant of the sample
(20 μL) was used as a quality control (QC).

[19]
As described in a previous study , UPLC-MS/MS analyzers were carried out with a Thermo UHPLC-Q
Exactive HF-X system equipped with an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, i.d.,
1.7 μm; Waters, Milford, USA). Non-targeted metabolomics was conducted by Majorbio Bio-Pharm
Technology Co. Ltd. (Shanghai, China). In brief, the mobile phase A was 2% acetonitrile-water (containing
0.1% formic acid) and B was acetonitrile (containing 0.1% formic acid). The injection volume was 3 μL. The
flow rate was 0.40 mL/min and the column temperature was 40 °C. The mobile phase elution gradient is
presented in Supplementary Table 1. Mass spectrometric data were collected using an electrospray
ionization (ESI) source operating in positive and negative modes. Optimal conditions were set as follows:
heater temperature at 450 °C, capillary temperature at 320 °C, sheath gas flow rate at 50 arb, Aux gas flow
rate at 13 arb, and ion-spray voltage floating (ISVF) at -3,000 V in negative mode and 3,500 V in positive
mode, respectively. The normalized collision energy was 20-40-60 V rolling for MS/MS. Full MS resolution
was 70,000 and MS/MS resolution was 17,500. Data acquisition was performed in Data Dependent
Acquisition (DDA) mode. Detection was performed over a mass range of 70-1,050 m/z.

Raw LC/MS data were pre-processed using Progenesis QI software (Waters Corporation, Milford, USA).
The data matrix was pre-processed to retain at least 80% of the metabolic signatures detected in each
sample. Raw and annotated data for metabolomics are reported in Supplementary Table 2. Then, the
specific sample was estimated as the minimum metabolite value, and each metabolic signature was
normalized to the sum. Metabolites were then identified by searching databases [HMDB (http://www.hmdb.
ca/), Metlin (https://metlin.scripps.edu/), and Majorbio (https://cloud.majorbio.com)]. Meanwhile, the
variables of the QC samples with relative standard deviation (RSD) > 30% were excluded and converted to
log10 values to obtain final data matrix for subsequent analysis. Differential metabolites between the two
groups were mapped to their biochemical pathways by metabolic enrichment and pathway analysis based
on the KEGG database (http://www.genome.jp/kegg/). Statistical analysis was performed using Python
package “scipy.stats” (https://docs.scipy.org/doc/scipy/) to obtain the most relevant biological pathways for
experimental groups.

Metagenomic analysis
Metagenomic sequencing was performed based on previously published methods . The fermented liquid
[20]
was centrifuged for 10 min (4 °C, 10,000 g) to enrich microorganisms. The total microbial genomic DNA of
the samples was extracted using E.Z.N.A® Soil DNA Kit (Omega Biotek, USA), and then the quantity and
quality of extracted DNAs were determined. Total DNA was fragmented using Covaris M220 and 400 bp
TM
fragments were screened. The paired-end library was constructed using NEXTFLEX Rapid DNA-Seq