Impact of Freezing and Freeze Drying on Lactobacillus rhamnosus GG Survival: Mechanisms of Cell Damage and the Role of Pre-Freezing Conditions and Cryoprotectants

Abstract

Lactobacillus rhamnosus GG (LGG) is a common lactic acid bacteria used in the food industry with proven health benefits. Maintaining a high viability of probiotics during freeze drying and storage is crucial for their efficacy. The involvement of protectants and the optimization of operating conditions are promising techniques utilized to help bacteria microorganisms overcome environmental challenges. Although numerous studies have investigated the effectiveness of various protective agents in mitigating environmental stresses on bacterial cells and improving their survival during freeze drying, there is limited understanding of how freezing parameters impact the process by influencing ice crystal formation and bacterial cell microstructure. Therefore, this study systematically evaluates the effects of freeze-thawing and freeze-drying processes on the survival and metabolic activity of LGG. The results reveal that cell damage during freezing and freeze drying is a complex process influenced by a variety of physicochemical factors, including freezing conditions, sublimation and thawing processes, as well as the choice of cryoprotectants and reconstitution medium. Notably, freezing with water in liquid nitrogen at −196 °C resulted in the highest bacterial survival rate (90.94%) under short freezing durations, demonstrating the importance of freezing conditions. Freeze drying further reduced viability, with survival rates dropping to as low as 2% under suboptimal conditions. Interestingly, phosphate-buffered saline as a resuspension medium significantly increased the loss of viable LGG during both freezing and freeze drying. The addition of trehalose and skim milk as cryoprotectants enhanced survival to 15.17% post-freeze drying, emphasizing the role of protective agents in improving viability. This study provides novel insights into the critical role of freezing parameters and operational conditions in preserving probiotic viability, offering valuable guidelines for optimizing the freeze-drying process to maintain the functionality of probiotics.