Optimization and multi-modal mechanistic elucidation of a composite cryoprotectants system for freeze-drying of Staphylococcus saprophyticus HS-42 and Meyerozyma guilliermondii LS-76 isolated from Xuanwei ham

Staphylococcus saprophyticus HS-42 and Meyerozyma guilliermondii LS-76 are promising starter strains isolated from Xuanwei ham, but their viability is greatly reduced during vacuum freeze-drying. This study optimized composite cryoprotectant formulations and investigated the underlying protective mechanisms. Single-factor screening followed by an L9(34) orthogonal design and storage stability was employed to determine the optimal formulations. For S. saprophyticus HS-42 starter, the optimal cryoprotectant consisted of 3% sucrose, 7% lactose, 5% maltose, and 5% skim milk powder, resulting in a freeze-drying survival rate of 93.10 ± 2.01%. For M. guilliermondii LS-76 and the mixed-strain starter at a 3:2 wet weight ratio, the optimal formulation contained 3% sucrose, 5% lactose, 5% maltose, and 5% skim milk powder, with survival rates of 91.96 ± 1.64% and 92.02 ± 1.33%, respectively. FTIR, SYTO 9/PI staining, and SEM analyses revealed that the composite cryoprotectants improved freeze-drying tolerance possibly by forming a glassy matrix, stabilizing protein secondary structures, and maintaining cell membrane integrity. These findings provide a practical basis for the preparation of Xuanwei ham starter cultures.