Hypothesis

Personalized prebiotic supplementation designed to amplify endogenous GABA‑producing bacteria, as identified by fecal metagenomic quantification of glutamate decarboxylase (GAD) genes, will lead to a statistically significant greater reduction in anxiety symptoms (measured by GAD‑7) after 8 weeks compared with a standard dose of the GABA‑producing probiotic Lactiplantibacillus plantarum Lp815, and this effect will be mediated by increased fecal and plasma GABA concentrations.

Rationale

• The probiotic trial shows that exogenous Lp815 raises urinary GABA and improves anxiety scores, but relies on transient colonization of an introduced strain [1].
• Endogenous GABA‑producing microbes (e.g., certain Lactobacillus, Bifidobacterium, and Bacteroides species) possess GAD pathways that can be up‑regulated by specific fermentable substrates such as galacto‑oligosaccharides (GOS) or fructooligosaccharides (FOS).
• Metagenomic profiling of GAD abundance provides a functional biomarker that predicts which individuals harbor sufficient capacity for GABA synthesis when supplied with the appropriate prebiotic.
• Amplifying resident strains may yield more stable, long‑term GABA production than short‑lived probiotic engraftment, thereby enhancing downstream vagal signaling and central GABAergic tone.

Predictions

1. Participants with baseline fecal GAD gene copies above the cohort median will show a >30 % reduction in GAD‑7 scores after receiving the personalized prebiotic, whereas those below the median will show minimal change.
2. The personalized prebiotic group will exhibit a greater increase in fecal GABA (µmol/g) and plasma GABA (nM) than the probiotic group, correlating with symptom improvement.
3. Changes in GABA levels will mediate the relationship between prebiotic treatment and anxiety reduction, as demonstrated by mediation analysis.
4. Microbiome shifts will show enrichment of the targeted GAD‑positive taxa without significant alterations in overall alpha diversity, indicating a precise ecological effect.

Experimental Design

• Design: Double‑blind, randomized, three‑arm parallel trial (n = 180, 60 per arm).
• Arms: (A) Personalized prebiotic (GOS/FOS blend) dosed according to baseline GAD abundance; (B) Standard probiotic Lp815 × 10⁹ CFU daily; (C) Placebo maltodextrin.
• Duration: 8 weeks supplementation + 4‑week follow‑up.
• Measurements: Baseline and weekly fecal metagenomics (shotgun) for GAD gene abundance; fecal and plasma GABA via LC‑MS; GAD‑7 and ISI questionnaires; adverse event monitoring.
• Analysis: Primary outcome – change in GAD‑7 score at week 8; ANCOVA adjusting for baseline. Secondary – mediation of GABA levels. Subgroup analysis by high vs low baseline GAD.

Falsifiability

If the personalized prebiotic fails to produce a significantly greater improvement in GAD‑7 scores than probiotic or placebo, or if fecal/plasma GABA does not rise disproportionately in responders, the hypothesis is refuted. Likewise, a lack of correlation between baseline GAD abundance and treatment response would invalidate the biomarker approach.