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Tian et al. Microbiome Res Rep 2024;3:49 https://dx.doi.org/10.20517/mrr.2024.33 Page 5 of 14

The Gastrointestinal Symptom Rating Scale (GSRS), developed by Svedlund et al., was used for this
[24]
purpose . The scale items were classified into “Bowel dysfunction syndrome”, “Indigestion syndrome”,
“Dyspeptic syndrome”, and “Abdominal pain syndrome”. GSRS scores were generated from self-evaluation
based on experiences during the past week.

Fecal 16S rDNA amplicon sequencing analysis
The genomic DNA of the samples was PCR-amplified using primers targeting the V3-V4 region of bacterial
16S rDNA (341F: CCTAYGGGRBGCASCAG, 806R: GGACTACNNGGGTATCTAAT, with a 7-base
barcode appended to the 5’ end of 341F to distinguish different samples). The PCR reaction system (50 µL)
comprised Taq Plus master mix (Jiangsu Kangwei Century Biotechnology Co., Ltd.), primers (0.8 µM each),
template DNA (2 µL), and nuclease-free water (added to make up the reaction volume to 50 µL). The PCR
program included Step 1: 95 °C (3 min); Step 2: 95 °C (30 s), 52 °C (30 s), 72 °C (30 s), for 30 cycles; Step 3:
72 °C (6 min), 4 °C (4 min). Following PCR amplification, the products were subjected to 1.5% agarose gel
electrophoresis (100 V, 100 mA, 30 min), and the bands of interest were purified to obtain the genomic
DNA samples of intestinal bacteria from mice. The purity and concentration of the samples were assessed
using Nanodrop, and a 50 µL mixed library with equal concentrations of all samples was constructed.
Sequencing was performed using the Illumina MiSeq platform (Illumina, USA).

Fecal untargeted metabolomics analysis
Accurately weigh 30-50 mg of vacuum freeze-dried fecal samples, and add 1 mL of methanol-water solution
(methanol: water, 9:1, v/v) for homogenization. Incubate the mixture at -20 °C for 1 h to precipitate
proteins, then centrifuge the mixture (15,000 g, 4 °C, 15 min). Concentrate the supernatant to dryness under
vacuum, resuspend in 100 μL of methanol-water solution, and then centrifuge. The supernatant is filtered
through a 0.22 μm organic filter membrane and subjected to liquid chromatography-mass spectrometry
(LC-MS) analysis. Statistical analysis of the data was performed using MetaboAnalyst 5.0 software (http://
www.metaboanalyst.ca/).

Fecal lactic acid content analysis
Fecal samples from the population were freeze-dried to constant weight. Equal portions of 50 mg freeze-
dried samples were transferred to 10 mL centrifuge tubes, and 300 μL of distilled water was added. After
thorough mixing, homogenization was performed using a homogenizer. Subsequently, 1.2 mL of 10%
chloroform was added to precipitate proteins. After centrifugation for 15 min (15,000 g, 4 °C), the
supernatant was aspirated using a disposable sterile syringe, filtered through a 0.22 μm organic filter
membrane, and transferred to 1.50 mL sample bottles for analysis using LC-MS. The concentration of
metabolites was quantified based on a standard curve.

Statistical analysis
Numeric results were presented as “mean ± standard deviation”. Data analysis and graph plotting were
performed using Prism 9.0. Differences before (pre) and after (post) placebo or probiotic intervention were
analyzed using paired t-tests. Comparisons of score changes between the probiotic and placebo groups were
conducted using unpaired t-tests. P-values for multiple comparisons were adjusted using family-wise
significance, and statistical significance was considered at a 95% confidence interval with a P < 0.05 in all
comparisons.

RESULTS
CCFM6432 improves mood, sleep, and gastrointestinal function in patients with depression
Under ethical requirements, all patients continued to take antidepressant medications without interruption
during the trial (see Table 1 for medication and demographic information). Therefore, to assess the effect of