The investigation of the mechanism underlying variations in oxidative stress tolerance of Lacticaseibacillus paracasei resulting from fermentation methods through endogenous CRISPR-Cas9 editing methodology.

The probiotic effects of lactic acid bacteria make them widely used in human and animal breeding industry. However, the presence of oxidative stress during the production and application process can cause bacterial damage or even death, significantly compromising the functionality of probiotics. Despite its potential for broader application scenarios that could provide a more comprehensive understanding of bacteria's internal adaptation strategies, there is a lack of research investigating oxidative stress from the perspective of culture methods. In this study, the tolerance to oxidative stress was compared between bacteria cultivated through solid-state fermentation (SSF) and liquid-state fermentation (LSF), and the physiological and transcriptional disparities between these two bacterial strains were investigated. Additionally, a novel and efficient gene editing method was developed to elucidate the genetic basis underlying these differences in tolerance. The results demonstrated a significantly higher tolerance to oxidative stress in SSF bacteria compared to LSF bacteria, along with a stronger capacity for maintaining intracellular microenvironment stability and the activity of key metabolic enzymes. It is noteworthy that the bacteria from SSF significantly enhance the transport of carbohydrate substances and facilitate intracellular metabolic flow. Gene editing experiments have confirmed the crucial role of genes glpF and glpO in regulating the glycerol metabolism pathway, which is essential for enhancing the tolerance of bacteria from SSF to oxidative stress. Based on these findings, the mechanism underlying the disparity in oxidative stress tolerance resulting from different culture methods has been summarized. Furthermore, investigation into different culture modes has revealed that moderate oxygen levels during cultivation significantly influence variation in bacterial tolerance to oxidative stress. Importantly, these variations are species-specific and depend on the ecological niche distribution of Lactobacilli. These findings elucidate a novel mechanism by which Lacticaseibacillus paracasei Zhang tolerates oxidative stress, and also suggest that distinct cultivation and processing methods should be tailored based on the specific Lactobacilli groups to achieve optimal application effects in production.