Development and Mechanistic Insights into Lyoprotective Technology for Akkermansia muciniphila Live Biotherapeutic Products

To explore the optimal combination of lyoprotective agents for effectively enhancing the viability of Akkermansia muciniphila (Akk) and elucidating the underlying mechanisms.This study aimed to optimize lyoprotectant formulations using single-factor experiments and response surface methodology for freeze-drying (lyophilization) of Akk DSM 22959. Bacterial powder was analyzed using Fourier-transform infrared spectroscopy (FTIR), glass transition temperature (Tg), and scanning electron microscopy (SEM) and by assessing Na⁺/K⁺ ATPase activity and membrane integrity. The formulation sustained Na⁺/K⁺ ATPase activit and achieved a post-lyophilization viability of 83.41%±0.41%. The survival rate of Akk powder was still higher after storage at −20 ℃ and 4 ℃ for 60 days. The optimal lyoprotectant (composed of skim milk powder, maltodextrin, sodium glutamate, and tremella polysaccharides) formed a protective matrix that encapsulated the Akk cells. This matrix preserved membrane integrity, maintained physiological morphology, and elevated Tg, thereby substantially reducing cryo-injury during lyophilization.These findings provide critical mechanistic insights into lyoprotection strategies and establish a scalable protocol for industrial production of live Akk biotherapeutic products.