Specific Lactobacillus strains improve anxiety symptoms by enhancing vagal afferent signaling through enterochromaffin cell serotonin release driven by microbial SCFAs.

Hypothesis Supplementation with Lactobacillus rhamnosus DM163 at 6×10^10 CFU daily will produce a measurable increase in HRV RMSSD within 10 days, preceding any significant reduction in self‑reported anxiety scores by at least 14 days. This HRV shift will depend on intact vagal integrity and will be abolished by acute pharmacological vagal blockade, while luminal SCFA concentrations remain unchanged.

Rationale L. rhamnosus DM163 modulates GABAergic and BDNF pathways in the brain, effects that disappear after vagotomy in rodents【3】. SCFAs produced by Lactobacillus stimulate enterochromaffin cells to release serotonin, which activates vagal afferents via 5‑HT3 receptors【4】. Increased serotonergic vagal firing enhances parasympathetic outflow, raising HRV indices such as RMSSD before central mood circuits are altered. Thus, HRV offers a real‑time readout of the gut‑brain axis that mirrors the mechanistic cascade from microbial metabolites to vagal tone to anxiety relief.

Predictions

1. In a double‑blind, placebo‑controlled crossover trial (n = 30 healthy adults with moderate stress), the L. rhamnosus DM163 group will show a ≥10 % rise in RMSSD averaged over days 8‑12 relative to baseline (p < 0.05).
2. STAI‑State scores will not differ significantly between groups until day 22, confirming a temporal lag.
3. Administering a short‑acting vagal antagonist (e.g., low‑dose atropine) on day 10 will blunt the HRV increase without affecting fecal acetate, propionate, or butyrate levels measured concurrently.
4. The placebo group will exhibit no HRV change, and L. delbrueckii control strain will fail to elicit either HRV or anxiety improvements, reinforcing strain specificity.

Methods Overview

• Participants receive capsules containing either L. rhamnosus DM163 (6×10^10 CFU) or maltodextrin placebo for 28 days, with a 2‑week washout before crossover.
• HRV is recorded each morning using a validated chest strap; RMSSD and SDNN are extracted via Kubios.
• Anxiety is assessed weekly with STAI‑State and DASS‑21.
• Fecal SCFAs are quantified by GC‑MS on days 0, 10, 20, 28.
• On day 10 of each arm, a subset receives atropine (0.01 mg/kg IV) or saline in a double‑blind fashion to test vagal dependence.

Falsifiability If RMSSD does not rise significantly before anxiety improvement, or if vagal blockade fails to attenuate the HRV response while leaving SCFAs unchanged, the hypothesis is refuted. Likewise, a lack of strain‑specific effects (i.e., L. delbrueckii producing similar HRV shifts) would undermine the proposed mechanistic link.

Impact Confirming HRV as an early, vagally mediated biomarker would accelerate probiotic trial design, reduce reliance on subjective endpoints, and clarify dose‑response and strain‑selection criteria for psychobiotic interventions.