Hypothesis

Baseline fecal short‑chain fatty acid (SCFA) profile and Bifidobacterium abundance can guide the selection of a personalized prebiotic (GOS for low Bifidobacterium, resistant starch for low Lactobacillus) that will significantly increase colonic butyrate production, which in turn raises central GABA levels via vagal afferent signaling and HDAC inhibition, leading to a clinically meaningful reduction in anxiety symptoms compared with non‑targeted prebiotic or placebo.

Rationale

While non‑targeted prebiotics show only a nonsignificant trend toward depression improvement (SMD: –0.28)【https://academic.oup.com/nutritionreviews/article/83/7/e1504/7934047】, probiotic strains such as Lactiplantibacillus plantarum Lp815 reduce anxiety by boosting urinary GABA【https://doi.org/10.1101/2025.04.14.25325830】. The mechanistic link between gut‑derived butyrate and GABAergic tone is supported by evidence that butyrate crosses the blood‑brain barrier, inhibits histone deacetylases, and up‑regulates glutamic acid decarboxylase (GAD) expression in enteric neurons and the hypothalamus【https://pmc.ncbi.nlm.nih.gov/articles/PMC12585160/】. Moreover, butyrate stimulates afferent fibers of the vagus nerve, which relay signals to the nucleus tractus solitarius and subsequently modulate limbic GABAergic circuits【https://biomedres.us/pdfs/BJSTR.MS.ID.009477.pdf】. Therefore, a precision approach that enriches butyrate‑producing taxa should amplify this pathway beyond the modest effects seen with indiscriminate prebiotics.

Predictions

1. Participants receiving the microbiota‑matched prebiotic will exhibit a ≥30 % increase in fecal butyrate concentration after 4 weeks relative to baseline.
2. Urinary GABA excretion will rise proportionally to butyrate increase, mirroring the GABA‑probiotic effect observed with Lp815【https://doi.org/10.1101/2025.04.14.25325830】.
3. Anxiety scores (GAD‑7) will decrease by ≥5 points (clinically significant) in the precision prebiotic arm, outperforming both the non‑targeted prebiotic and placebo groups.
4. Changes in anxiety will mediate (account for >40 % of) the effect of butyrate elevation on symptom improvement, as tested by mediation analysis.

Experimental Design

• Population: 150 adults with moderate anxiety (GAD‑7 ≥10) and no current psychotropic medication.
• Stratification: Baseline shotgun metagenomics and targeted SCFA quantification (GC‑MS) to classify participants as Bifidobacterium‑low, Lactobacillus‑low, or mixed deficiency.
• Intervention Arms (double‑blind, 8 weeks):
  1. Precision prebiotic: 'GOS' (5 g/day) for Bifidobacterium‑low; resistant starch (10 g/day) for Lactobacillus‑low; combined dose for mixed deficiency.
  2. Non‑targeted prebiotic: inulin‑FOS blend (10 g/day) identical to prior RCTs【https://academic.oup.com/nutritionreviews/article/83/7/e1504/7934047】.
  3. Placebo: maltodextrin.
• Outcomes:
  • Primary: change in GAD‑7 score.
  • Secondary: fecal butyrate, acetate, propionate (GC‑MS); urinary GABA (LC‑MS/MS); microbiota composition (16S rRNA shotgun); cytokine panel (IL‑6, TNF‑α).
• Statistical Plan: ANCOVA adjusting for baseline scores, mediation analysis (butyrate → urinary GABA → anxiety), correction for multiple testing (FDR <0.05).