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

comprehensively evaluate the adjunctive therapeutic effect of Pediococcus acidilactici CCFM6432 on
depressive symptoms through a randomized, placebo-controlled clinical trial and to explore the potential
material basis for its efficacy.

METHODS
Probiotic strain and placebo
Pediococcus acidilactici CCFM6432 was obtained from the Culture Collection of Food Microorganisms
(CCFM) at Jiangnan University. The probiotic formulation used in this trial consisted of freeze-dried
powder of CCFM6432 and maltodextrin. The specific preparation process is as follows: After two successive
activations on MRS solid medium (37 °C, cultured for 36-48 h), a single colony was picked and inoculated
into MRS liquid medium. After incubating at 37 °C for 18 h, the culture was further propagated at the same
conditions with a 1% (v/v) inoculum to increase biomass. A 2% (v/v) inoculum was then fermented in a 20-
liter fermenter for 18 h. The fermentation broth was centrifuged to obtain bacterial slurry. Subsequently, the
bacterial slurry was mixed with protectants at a ratio of 1:5 (w/v) (protectants composed of 13% skim milk,
3% sucrose, and 3% inulin) and freeze-dried to obtain the probiotic powder of CCFM6432. The probiotic
powder was then packed into sachets (2 g/sachet, with a viable count of ≥ 10 CFU/sachet). Maltodextrin
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placebo was prepared in the same manner without the addition of bacterial powder.

Clinical trial design
This clinical trial is a randomized, placebo-controlled study. The study was conducted collaboratively by the
Food Biotechnology Center of Jiangnan University and Yixing People’s Hospital. The clinical trial design
complies with the Helsinki Declaration and has been approved by the Medical Ethics Committee of Yixing
People’s Hospital (WXMHCIRB2023LLky002, Yixing, China). Clinical Trial Registration Number:
ChiCTR2300071025.

In this study, we used G Power software to calculate the required sample size for participant recruitment,
using the methodology from a previous study by Majeed et al. as a reference . Specifically, in their study
[20]
on the effects of probiotics on alleviating depression, the Hospital Anxiety and Depression Scale (HADS)
scores were 13.60 ± 4.41 (Mean ± SD) before treatment and 5.90 ± 4.88 (Mean ± SD) after treatment. Using
an alpha level of 0.05, a power (1-β) of 0.80, and an effect size of 0.94, we estimated the minimum total
sample size to be 38 participants, with an actual power of 0.80. Consequently, at least 19 patients were
required per group.

Inclusion criteria: (1) Clinical diagnosis of depressive disorder or anxiety disorder; (2) HADS score > 11; (3)
Aged 18 years and above; (4) Primary school education or above, without speech expression disorders; (5)
Subject or guardian has signed an informed consent form; (6) Subject fills out a written informed consent
form, basic information questionnaire, dietary structure and lifestyle questionnaire, and commits to
cooperate with the study throughout the entire trial period. Exclusion criteria: (1) Treatment-resistant
depression; (2) Meets DSM-IV Axis I diagnosis of other mental disorders; (3) Suicidal or self-harming
behavior; (4) Patients with schizophrenia, bipolar disorder, neurodegenerative diseases; (5) Severe physical
illnesses such as HIV, history of epilepsy, history of heart disease, hyperthyroidism, and other serious
physical illnesses; (6) Pregnant or lactating women; men or women during preconception (conception); (7)
Use of antibiotics, probiotics, and their products in the past month.

The entire intervention period lasted for four weeks. Assessments were conducted at the beginning and end
of the trial, including questionnaire evaluations, and serum and fecal sample collection. Throughout the
trial, participants in the placebo control group were required to take 1 sachet of placebo (maltodextrin)