Optimized composite cryoprotectants enhance survival and membrane integrity of Bifidobacterium breve BX-18 during...

Bifidobacterium breve BX-18 is a probiotic strain with documented health benefits, yet lyophilization compromises its viability. We hypothesized that optimizing a composite cryoprotectant would enhance survival by preserving membrane integrity and reducing oxidative stress. Single-factor screening identified skim milk, trehalose, sodium acetate, and taurine as effective protectants. Response surface methodology (Box-Behnken design) determined the optimal formulation: trehalose (10.66%), sodium acetate (0.98%), taurine (0.42%), and skim milk (9.75%), achieving 71.01% survival versus <5% in unprotected controls. Mechanistic analyses revealed the composite protectant preserved membrane integrity (70.42% intact cells vs. 28.32% control), maintained membrane fluidity (generalized polarization 0.026 ± 0.011 vs. 0.047 ± 0.009 postlyophilization), and retained enzyme activities (Na⁺-K⁺-ATPase decreased 26.3% vs. 76.4% in controls, Ca²⁺/Mg²⁺-ATPase decreased 38.4% vs. 62.9%), and β-galactosidase activity remained higher (10.02 ± 0.15 U/mL vs. 2.62 ± 0.13 U/mL). Intracellular reactive oxygen species accumulation was significantly attenuated, and intracellular malondialdehyde content was significantly reduced, indicating alleviated lipid peroxidation in the protected group. Storage stability assessment further demonstrated that lyophilized B. breve BX-18 prepared with the optimal composite cryoprotectant maintained significantly higher viable counts during 21 d of refrigerated storage (4°C) in fermented milk, meeting the viable cell requirements for probiotic functional foods. These findings demonstrate that strategic cryoprotectant combinations preserve B. breve BX-18 viability through multilevel cellular protection, supporting the development of stable probiotic ingredients for functional dairy applications.