Changes in the metabolome of probiotics during the stationary phase increase resistance to lyophilization

In the probiotics manufacturing process, lyophilization damages the cell membrane of microorganisms, reducing their viability and delaying reactivation of their cellular metabolism. The growth phase of microorganisms at the time of lyophilization is thought to significantly affect their viability during storage and reactivation in the in-testine. However, no study has systematically investigated the differences in overcoming mechanisms in mi-croorganisms harvested at different growth phases against lyophilization. Therefore, in this study, we comparatively assessed the resistance mechanism of Bifidobacterium animalis ssp. lactis and Lacticaseibacillus rhamnosus harvested at exponential and stationary growth phases. We found that B. animalis ssp. lactis and L. rhamnosus harvested at the stationary phase had shorter lag phase in growth and higher survival rates during storage than those harvested at the exponential phase. As a result of metabolome analysis, the cells harvested at the stationary phase accumulated trehalose and arabinose, which function as cryoprotectants and protect cells from lyophilization-induced damage. These findings could provide a basis for establishing a manufacturing protocol for lyophilized probiotics that can be rapidly reactivated in the intestine to achieve optimal metabolic activity and induce health benefits.