Immunostimulatory effects of Bacillus subtilis-fermented garlic (Aglio): an in-depth in vitro and in vivo analysis

Abstract

This study evaluated the immunostimulatory potential of garlic fermented with Bacillus subtilis (Aglio) and identified the underlying mechanisms using in vitro and in vivo models. Aglio significantly enhanced macrophage activity, with increased TNF-α (9.3–46.6 fold), MCP-1 (5.3–41.4 fold), IL-6 (2.1–32.1 fold), and IL-12 (1.1–5.5 fold) secretion compared to those of the standard garlic extract. This macrophage-stimulatory activity was associated with MAPK (ERK, JNK, and p38) and NF-κB (IκBα and p65) signaling pathway activation. Aglio significantly increased splenocyte proliferation (1.8–2.9 fold) and TNF-α (32.5–96.6 fold), IFN-γ (26.6–362.3 fold), GM-CSF (2.1–3.9 fold), and IL-6 (10.3–11.6 fold) secretion. Gene expression analysis revealed Th1-related T-Bet upregulation and Th2- and Th17-related GATA3 and FOXP3 downregulation, indicating a Th1-mediated splenocyte activation mechanism. Oral administration of Aglio (125 and 250 mg kg⁻¹) to BALB/c mice increased splenocyte proliferation (2.1–3.3 fold) and elevated splenic cytokine (TNF-α, 1.9–2.7 fold; GM-CSF, 2.2–2.3 fold; IL-6, 1.9 fold) and antibody (IgA, 1.4–1.8 fold; IgG, 1.0–1.7 fold) levels. Aglio administration also increased serum TNF-α (2.1–3.3 fold), IL-6 (1.0–1.1 fold), and IgG (1.6–1.9 fold) levels. Nutrient analysis indicated that Aglio lacked detectable carbohydrates and had negligible protein and polyphenol contents compared to standard garlic extract, suggesting complete biotransformation during fermentation. These findings demonstrate Aglio-mediated immune activation, highlighting its potential as a functional food or nutraceutical agent for immune enhancement.