Effects Of Cross-stress On Freeze-drying Survival Rate And Stability During Room Temperature Storage Of Lactiplantibacillus Plantarum LIP-1

Proper stress pretreatment of probiotics can improve their resistance to adverse environment and help maintain the survival rate of probiotics during production and storage.At present,researches on improving the freeze-drying resistance and room temperature storage stability of probiotics have mostly focused on single stress treatment,few studies have focused on the impact of cross-stress on the survival rate of strains.In this thesis,a Lactiplantibacillus plantarum LIP-1 was selected as the research object,on the basis of determining the optimum pre-freezing temperature for freeze-drying based on the freeze-drying survival rate and stability during room temperature storage,the cells at different growth stages were treated with oxygen,cold and oxygen-cold cross-stress,respectively,to investigate the effects of different stress treatments on the freeze-drying survival rate of the strain and related mechanisms,the optimal group was taken as an example to explore the effects of stress treatment on the stability during room temperature storage of the strain and the internal mechanism.The main research results are as follows:（1）By exploring the effects of different pre-freezing temperatures on the freeze-drying survival rate and stability during room temperature storage of Lactiplantibacillus plantarum LIP-1,it was found that the freeze-drying survival rate and stability during room temperature storage of strain in-80℃group were significantly higher than those in-20℃group（P<0.05）.Exploring its internal mechanism and found that compared with the-20℃group,the-80℃group had a lower level of intracellular reactive oxygen species（ROS）,which reduced the oxidation degree of cell membrane fatty acids,effectively maintained the integrity and fluidity of cell membrane during freeze-drying and room temperature storage,and significantly improved the survival rate of the strain.Therefore,subsequent freeze-drying was carried out at the pre-freezing temperature of-80℃.（2）By exploring the effects of oxygen stress,cold stress and cross-stress on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1,it was found that compared with the control group without stress treatment,oxygen stress in the lag phase（2.6 mmol/L H₂O₂）,cold stress in the stationary phase（10℃,1.5 h）and two periods of oxygen-cold cross-stress could significantly improve the freezing-drying survival rate of the strain,and the cross-stress group had the best effect（P<0.05）.（3）Through the experiments,it was found that the internal mechanism of cross-stress treatment to improve the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1could be divided into the following two aspects:a.Reduced subcellular structural damage:Compared with single oxygen stress and cold stress,the cross-stress group increased the S-layer productiont of the strain,made the cell wall structure more stable.By enhancing the antioxidant capacity of the strain,oxidative damage caused by intracellular ROS on fatty acids of cell membrane was effectively reduced,protected the integrity and fluidity of cell membrane.By promoting the production of ATP,it provided energy for the strain to pump proton（H⁺）out of the cell,alleviated the damage caused by low acid environment on DNA,and maintained the integrity of DNA structure.b.Promoted biofilm synthesis and stress proteins production:Compared with the single oxygen stress and cold stress treatment,the cross-stress group had a higher ability to promote the synthesis of biofilms of the strain,established a protective layer outside the cell,and enhanced the strain’s own stress resistance.At the same time,it could promote the strain to produce more stress proteins,which improved the strain’s tolerance to adverse environment.（4）By exploring the effects of cross-stress on stability during room temperature storage of Lactiplantibacillus plantarum LIP-1,it was found that compared with the control group without stress treatment,on the one hand,under the effect of cold stress,the cross-stress group improved the integrity and compactness of cell membrane by adjusting the relative content of unsaturated fatty acids in cell membrane and promoting the formation of long chain unsaturated fatty acids.On the other hand,under the effect of oxygen stress,the cross-stress group could better maintain the antioxidant properties of the strain by reducing the loss of SOD activity in the cell,slow down the oxidation rate and degree of fatty acids oxidation of the cell membrane,so as to maintain the integrity and fluidity of the cell membrane,and thus significantly improved the stability during room temperature storage of the strain.In conclusion,this study was the first time to explore the effects of oxygen-cold cross-stress on the freeze-drying survival rate and stability during room temperature storage of the strain,and revealed its internal mechanism.This study provides new ideas and theoretical support for solving the bottleneck problem of probiotic industry development.