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Tian et al. Microbiome Res Rep 2024;3:49 https://dx.doi.org/10.20517/mrr.2024.33 Page 9 of 14

significant inter-group differences in neural system-related signaling pathways [Figure 3G].

Positive correlation between increased intestinal lactic acid levels mediated by probiotics and
improvement in patient mood
Considering the alterations in specific gut microbial taxa abundance in patients with depression following
probiotic intervention, it was hypothesized that this might lead to significant changes in the spectrum of
intestinal metabolites. To investigate this, a non-targeted metabolomic approach based on LC-MS was
employed to detect the metabolite composition of fecal samples from patients with depression. The
obtained data were compared and annotated against the Mzcloud and Kyoto Encyclopedia of Genes and
Genomes (KEGG) databases, with substances matched with a score greater than 80 being selected.
Ultimately, 137 substances were identified in positive ion mode and 257 substances in negative ion mode.

To further explore key biomarkers before and after CCFM6432 intervention, a volcano plot was used to
display metabolites with fold change values greater than 2 and p-values less than 0.05 (t-test) between
groups. Compared to pre-CCFM6432 intervention or post-placebo intervention, the levels of DL-lactic acid,
GABA, N-acetyl-L-phenylalanine, and N-acetylvaline in the intestine were significantly elevated after
CCFM6432 intervention [Figure 4A and B]. Among these substances, lactic acid exhibited the most
significant inter-group differences. Furthermore, absolute quantification of lactic acid in feces using LC-MS
validation confirmed that the intake of CCFM6432 significantly increased the level of lactic acid in the
intestines of patients with depression, with a significantly higher increase than the difference before and
after placebo treatment [Figure 4C]. Finally, for the differential metabolites identified in Figure 4A and B,
debias sparse partial correlation (DSPC) network analysis was conducted to assess the correlation between
changes in metabolite levels and changes in mood, sleep, and gastrointestinal scale scores [Figure 4D]. It
was found that there was a significant negative correlation between the change in fecal lactic acid content
and the change in HADS score (i.e., higher lactic acid levels were associated with lower HADS scores;
Partial Coeff. = -1, P = 0.024); however, the change in lactic acid levels was negatively correlated with
changes in PSQI and GSRS scores, but without statistical significance (Partial Coeff. = -1, P = 0.154; Partial
Coeff. = -0.363, P = 0.179, respectively). Additionally, a significant negative correlation was observed
between the change in N-Acetyl-L-phenylalanine content and the change in HADS score
(Partial Coeff. = -1, P = 0.046).

DISCUSSION
In this study, we evaluated the effects of Pediococcus acidilactici CCFM6432 on emotional performance,
gastrointestinal function, gut microbiota, and the distribution of their metabolic products in patients with
depression. Although the intake of this strain did not significantly alter the overall structure of the gut
microbiota (beta diversity) in patients, Pediococcus acidilactici CCFM6432 demonstrated good colonization
characteristics, becoming a distinguishing species between the gut microbiota of the two groups. Notably, it
significantly increased the lactic acid content in the gut, and this change was positively correlated with
improvements in mood, gastrointestinal function, and sleep quality. Specifically, we observed a significant
reduction in the annotation abundance of the NOD-like receptor signaling pathway in the gut microbiome.
Given the well-known antibacterial effects of lactic acid, the brain-beneficial functions of Pediococcus
acidilactici may be associated with the inhibition of inflammation signaling pathways mediated by
microorganisms.

Over the years, the role of the immune system has been closely linked to the etiology and pathophysiology
of depression . For instance, clinical studies have found significantly elevated levels of pro-inflammatory
[32]
cytokines, such as interleukin-6 (IL-6), TNF-α, and C-reactive protein, in the serum or cerebrospinal fluid of