Study On Antibacterial Activity And Mechanism Of Rhamnolipids Against Bacillus Cereus

Bacillus cereus is a common food-borne pathogenic bacterium.It usually presents in the food processing environment in three forms: nutrient cells,spores and biofilm,causing serious economic losses and negative impacts to the relevant processing companies as a result of food contamination.The addition of chemical synthetic food preservatives is a commonly used method for prevention and control of foodborne pathogens such as Bacillus cereus,but is not in line with current food consumption concepts such as green and safe.Therefore,it is important to explore new natural food antimicrobial agents for the prevention and control of different forms of Bacillus cereus contamination.Rhamnolipids(RLs)are non-toxic glycolipid biosurfactants with promising applications in the antibacterial field,but the antibacterial activity and mechanism of different forms of Bacillus cereus have been less studied,limiting their own application in the food industry.This thesis firstly investigated the antibacterial activity and mechanism of rhamnolipids against nutrient cells and spores of Bacillus cereus,and applied RLs to the preparation of fresh wet noodles to evaluate the antibacterial activity and the effect on product quality in food.On this basis,we investigated the inhibitory effect of RLs on formation and removal of Bacillus cereus biofilm,focusing on the removal effect and the mechanism of influence on biofilms in combination with ultrasound,with the following main findings:(1)RLs showed good antibacterial activity against Bacillus cereus in the trophic state,where the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)were 16.0 mg/L and 32.0 mg/L,respectively.Under the treatment of RLs of MIC,the surface of the bacterium appeared wrinkled and depressed,and the integrity of the cell membrane was disrupted.At the same time,RLs increased the permeability of cell membranes,prompting a massive leakage of intracellular macromolecules,reducing the metabolic activity of bacterium and causing oxidative stress in the bacteriophage,resulting in multiple damages.Experiments on the preparation and storage of fresh wet noodles revealed that RLs had a significant killing effect on Bacillus cereus in fresh wet noodles,with a sustained inhibition effect at higher concentrations.At the same time,RLs promoted the conversion of free water to bound water in fresh wet noodles,resulting in less microbially available water,which helped the storage of fresh wet noodles and gave it a tougher texture and better chewiness.(2)The MIC and MBC of RLs against Bacillus cereus spores were 80.0 mg/L and 160.0mg/L respectively.Microscopic studies have revealed RLs cause severe wrinkling and depression of the budding surface,disrupting structural integrity and permeability of spores,leading to the leakage of intracellular biomolecules such as electrolytes,DNA and proteins.At the same time,RLs inhibited and inactivated spores through a variety of antimicrobial mechanisms such as reducing heat resistance,generating intracellular oxidative stress,reducing surface adhesion,and binding and interfering with DNA molecules.(3)The minimum biofilm inhibitory concentration(MBIC)of RLs against Bacillus cereus was 32.0 mg/L,while the removal of mature biofilm needed to be improved.Combined ultrasound(US)treatment was found to significantly improve the clearance efficiency of RLs,clearance of 32.0 mg/L RLs + US 20 min on the biofilm largely consistent with 256.0 mg/L RLs alone,indicating a synergistic antibacterial effect of RLs and ultrasound.To characterize the effects of RLs and US treatments on bacterial cells and extracellular matrix in biofilm,it was found that the metabolic activity,surface hydrophobicity and motile aggregation ability of bacterial cells were significantly reduced,the synthesis and secretion of polysaccharide,protein and eDNA in extracellular matrix were also significantly reduced.The formation process of biofilm was seriously damaged.