Accumulation of K ⁺ and glutamate inside the cell and unsaturated fatty acid content in cell membranes contribute to osmotic stress tolerance in <em>Bifidobacterium bifidum</em>

<em>Bifidobacterium bifidum</em>, a key commensal promoting gastrointestinal health and immune function faces industrial challenges due to high osmotic pressure from acid accumulation during fermentation and water loss during freeze-drying. This study identified the strain <em>B. bifidum</em> CCFM1301 with superior osmotic pressure tolerance among 85 strains, using <em>B. bifidum</em> CCFM16 as a control. Inductively coupled plasma - mass spectrometry (ICP-MS) results indicated that <em>B. bifidum</em> CCFM1301 accumulated K⁺ to combat osmotic stress, while gas chromatography-mass spectrometry detection (GC-MS) analysis revealed an increase in unsaturated fatty acids (such as cyclopropane fatty acid) in its cell membrane. Non-targeted metabolomics suggested that <em>B. bifidum</em> CCFM1301 enhanced citric acid metabolism (with a fold change (FC) of 21.07 for citric acid), supporting membrane fatty acid synthesis. In contrast, <em>B. bifidum</em> CCFM16, with lower osmotic pressure tolerance, accumulated intracellular glutamate (FC=14.34) under stress. The study confirmed that exogenous K⁺, glutamate, and unsaturated fatty acids (oleic acid) improved osmotic stress resistance, measured by osmotic pressure, viable bacterial count, and freeze-drying survival rate. This research enables high-density fermentation and improves freeze-drying survival rate of <em>B. bifidum</em>, providing a foundation for its industrial application.