Study On The Damage Mechanism And Protective Measures Of Lactate And Osmotic Stress On Lactobacillus Casei Zhang

In the fermentation industry of lactic acid bacteria,the production of a large amount of lactate salt and the formation of a high osmotic pressure environment after constant p H fermentation can cause damage to the bacterial body.However,the mechanism of the damage is not clear,and there is no good solution.Lacticaseibacillus casei Zhang（LCZ）has good fermentation characteristics and probiotic effects,and is widely used in industries such as food and medicinal health.In this study,LCZ was used as the research object,and three salt types,namely sodium lactate,ammonium lactate,and sodium chloride,were used to create fermentation stress environments to explore the mechanism of damage to bacterial activity caused by lactate salt stress and osmotic stress.At the same time,this study attempted to improve the fermentation activity and biomass of LCZ from the perspective of alleviating osmotic stress by selecting compatible solutes,optimizing the type of neutralizer,and optimizing the high-density fermentation mode,so as to provide technical support for the industrial production of strains.The specific research results are as follows:（1）The osmotic pressure conditions that inhibit the growth of LCZ and the salt concentration that inhibits its growth were tested.The results showed that LCZ began to be inhibited under an osmotic pressure of 1200 m Osm/kg,and its growth and reproduction were basically inhibited under an osmotic pressure of 3200 m Osm/kg.Different salt types exhibited different antibacterial properties to LCZ,with ammonium lactate having strong antibacterial properties and inhibiting completely at a concentration of 0.7mol/L,sodium lactate having mild antibacterial properties and inhibiting completely at a concentration of 1.4 mol/L,and sodium chloride exhibiting osmotic pressure inhibition and inhibiting completely at a concentration of 1.4mol/L.（2）The mechanism of damage to LCZ caused by lactate salt and osmotic stress was studied.The fermentation stress environment was created using sodium lactate,ammonium lactate,and sodium chloride at the concentration that completely inhibits LCZ.The techniques of plate counting,inhibition circle method,scanning electron microscopy,enzyme activity determination,flow cytometry,and fluorescence polarization were used to determine the damage to cell viability,cell morphology,K⁺Na⁺-ATPase enzyme activity,lactate dehydrogenase activity,intracellular and extracellular p H,glucose uptake capacity,and membrane fluidity.The results showed that the three salts caused damage to LCZ by inhibiting cell glucose uptake capacity and membrane fluidity,and inhibiting lactate dehydrogenase and K⁺Na⁺-ATPase enzyme activity.Ammonium lactate strongly inhibited LCZ’s cell glucose uptake capacity and membrane fluidity,while sodium lactate and sodium chloride had a lower inhibitory effect on cell glucose uptake capacity and membrane fluidity than ammonium lactate but higher than the control group.Lactate salt and osmotic stress had little effect on intracellular p H,and the change in intracellular p H was mainly related to extracellular p H.The inhibitory effect of the three salts on LCZ’s lactate dehydrogenase activity was in the order of ammonium lactate>sodium lactate>sodium chloride>control group,while the inhibitory effect of the three salts on LCZ’s K⁺Na⁺-ATPase enzyme activity was in the order of ammonium lactate>sodium lactate>sodium chloride>control group.（3）A fermentation regulation process was optimized and established.Based on the inhibition of lactate salt and osmotic pressure,the optimum fermentation regulation process included the use of a combined strategy of"calcium hydroxide+ammonia water"as a neutralizer in fermentation instead of ammonia water,and constant p H dilution feeding cultivation as a high-density fermentation mode.Under this process condition,the OD₆₀₀,dry weight yield,and live bacterial count of the fermentation were 20.192±0.094,1.735±0.077%,and（3.439±0.110）×10¹⁰CFU/m L,respectively,which were1.74,2.48,and 1.82 times higher than previous processes.This thesis systematically studied the mechanism of cell damage to Lacticaseibacillus casei Zhang under high-density fermentation conditions caused by lactate salt and osmotic stress,providing a theoretical basis for optimizing the fermentation process of lactic acid bacteria and improving the number and activity of live bacteria.