Hypothesis

Daily supplementation with Lactiplantibacillus plantarum LZU-J-TSL6 at 1×10⁹ CFU for six weeks will increase vagally mediated heart rate variability (RMSSD) and reduce self‑reported anxiety scores in healthy adults, and this autonomic shift will be mediated by increased hippocampal GABA/BDNF signaling driven by vagal afferent activation from gut‑derived serotonin and short‑chain fatty acids.

Rationale

• Pre‑clinical work shows L. rhamnosus JB‑1 requires an intact vagus to exert anxiolytic effects, linking probiotic action to vagal tone 2.
• L. plantarum LZU‑J‑TSL6 elevates hippocampal GABA, BDNF and 5‑HT while repairing the intestinal barrier 3.
• Heart rate variability, especially RMSSD, is a validated index of vagal efferent activity and predicts anxiety susceptibility 5.
• Gut microbes modulate enterochromaffin cell serotonin release and produce SCFAs that stimulate vagal afferents via 5‑HT3 receptors and free‑fatty‑acid receptors (FFAR2/3), providing a plausible mechanistic bridge from luminal changes to central neurotransmitter shifts.

Predictions

1. We'll measure a statistically significant rise in weekly RMSSD for the probiotic group compared with placebo, beginning at week two and peaking at week six.
2. It's expected that reductions in Perceived Stress Scale‑10 scores will correlate positively with RMSSD improvements (r > 0.4, p < 0.05).
3. Fecal concentrations of acetate, propionate and butyrate will increase in the probiotic group and mediate the RMSSD change (mediation analysis p < 0.05).
4. Plasma BDNF levels will rise in parallel with RMSSD, while hippocampal GABA measured by magnetic resonance spectroscopy will show a non‑significant trend toward increase due to measurement limits.
5. Administration of a selective vagal afferent blocker (e.g., low‑dose ondansetron to antagonize 5‑HT3) in a crossover sub‑study will attenuate the probiotic‑induced RMSSD rise, confirming vagal dependence.

Experimental Design

• Population: 80 healthy adults aged 21‑35, baseline RMSSD < 40 ms or PSS‑10 > 14, stratified by sex.
• Intervention: L. plantarum LZU‑J‑TSL6 1×10⁹ CFU in maltodextrin capsule daily vs identical placebo.
• Duration: 6 weeks supplementation plus 2‑week washout.
• Outcome Measures:
  • Primary: weekly 5‑minute resting ECG RMSSD.
  • Secondary: PSS‑10, fecal SCFA (GC‑MS), plasma BDNF (ELISA), optional hippocampal GABA (MRS subset n = 20).
• Statistical Plan: Mixed‑effects models with time, group, and interaction; mediation analysis for SCFA → RMSSD → anxiety; alpha = 0.05.

Potential Outcomes and Falsifiability

• Confirmatory: Significant group × time interaction for RMSSD, mediated by SCFA and BDNF, with vagal blockade reducing the effect → supports hypothesis.
• Null: No RMSSD difference between groups → refutes the claim that this strain modulates vagal tone sufficiently to impact autonomic HRV in humans.
• Alternative: RMSSD rises without anxiety change or without SCFA/BDNF shifts → suggests mechanisms beyond the proposed vagal‑GABA/BDNF axis, prompting revision of the mechanistic model.

This protocol directly translates the rodent vagal‑dependent anxiolytic pathway to a human autonomic readout, filling the translational gap highlighted by the lack of HRV validation for gut‑brain interventions 2.