Qula-derived Limosilactobacillus fermentum TD-3 and Lactococcus lactis MQ1-1 alleviate aging-related intestinal barrier dysfunction via microbiota–short-chain fatty acid–AMPK/MLCK–tight junction axis

The aging process is associated with detrimental effects on the integrity of the intestinal barrier, consequently elevating susceptibility to various diseases. Qula exhibits outstanding nutritional composition and has a characteristic taste profile, positioning it as a prime candidate for probiotic exploration given its rich reservoir of probiotic potential. In the present study, a probiotics screening strategy was developed based on the production of short-chain fatty acids (SCFA) to reverse aging-related intestinal barrier dysfunction. After screening, Limosilactobacillus fermentum TD-3 and Lactococcus lactis MQ1-1 isolated from traditional dairy product Qula were selected based on their acid and bile salt resistance, adhesion, and antibacterial properties. In the aging mice, the combination of probiotic mixture (PM) of Lb. fermentum TD-3 and Lc. lactis MQ1-1 enhanced intestinal barrier integrity by activating mitogen-activated protein kinase (AMPK) phosphorylation, which suppressed myosin light-chain kinase (MLCK) activity to increase the expression of tight junctions. Meanwhile, PM decreased colon oxidative stress (SOD, GSH-Px, and malondialdehyde) and inflammation (IL-6, IL-1β, TNFα, and IL-10), and restored the composition of the intestinal microbiota composition. Specifically, PM enriched the relative abundance of SCFAs producing bacteria, including g_ₙorankf_Muribaculaceae and g_Limosilactobacillus, g_Bifidobacterium, and g_Allobaculum, thereby increasing the acetic acid and butyric acid production. Thus, the combination of Lb. fermentum TD-3 and Lc. lactis MQ1-1 activated the AMPK signaling pathway by regulating the gut microbiota and metabolites. This study innovatively established a precision screening system for probiotics targeting SCFA, and isolated and identified a PM from traditional fermented dairy products (Qula) to relieve aging-associated intestinal barrier dysfunction.