Effects Of Ultrasonic Field Intensity On The Stress Response Of Pseudomonas Fluorescens Biofilm And Its Regulatory Strategy

Ultrasound can be used as a high-efficiency cleaning technology for edible agricultural products;it can also be used as a non-thermal sterilization method in food processing,which would maximize nutrients in foods and simultaneously ensure food safety.Pseudomonas fluorescens is a commonly appeared foodborne spoilage bacteria with a strong biofilm-forming ability.The P.fluorescens biofilm significantly enhanced its resistance to heat treatment and mechanical cleaning.Previous studies have shown that ultrasonic waves produced cavitation resulting in momentary high temperature and pressure,active free radicals,and strong shear stress.However,ultrasonic effects on biofilm removal and sterilization were weakened due to the rapid decay of sound waves in the cleaning medium and plant tissue.Meanwhile,ultrasound at a relatively low-intensity level would provide an ultrasonic stress to bacteria,which increased its resistance,significantly weakened the cleaning and sterilized effect,and eventually limited the application of ultrasonic technology in the field of processing edible agricultural products.Based on the above background,the effects of ultrasonic field intensity on the stress response and regulatory strategies of P.fluorescens in the biofilm state were investigated.Firstly,the effects of different intensity levels of ultrasound on the structure and function of P.fluorescens biofilm and quorum sensing were investigated.Results showed that ultrasound at relatively low-intensity levels induced stresses to P.fluorescens biofilm,which enhanced the motility of cells,secretion of exopolysaccharides,extracellular protease activity,and biofilm formation.Compared with the untreated group,the growth of P.fluorescens was significantly inhibited by high-intensity ultrasound with a reduced biofilm and inhibited quorum sensing.microscopy observations also supported the differences of morphology and biofilm structure undergone ultrasonic treatment at different intensity levels.Low-intensity ultrasound enhanced the synthesis of C8-HSL signaling molecule by 26.2%and the gene expression of pco I（2.16times）.Meanwhile,the expression levels of biofilm-related genes,i.e.flg A,alg D,apr X,and lap A were also significantly promoted;however,the expression levels of the above-mentioned genes were significantly down-regulated after the treatment of high-intensity ultrasound.Results of transcriptomic analysis on quorum sensing indicated that the low-intensity ultrasound activated the quorum sensing system of P.fluorescens and thereby,significantly enhanced its biofilm formation and resistance.Proteomic analysis was further conducted.Results showed that 183 differentially expressed proteins were expressed（p<0.05）after the ultrasonication.There were 164 and 19proteins that were up-regulated and down-regulated,respectively.Furthermore,the stress-responded mechanisms of P.fluorescens after the sonication was revealed:1)the proteins of flagellar assembly system were first activated after the sonication leading to changes in bacterial tropism;2)the ATP-binding cassette transporter accelerated the transmembrane transport of substances inside and outside the cell on the cell membrane and meanwhile,balancing the osmotic conditions on both sides of the cell membrane;3)the expression of proteins related to DNA repair was up-regulated after the treatment of ultrasound,which involved the repair of damaged DNA in P.fluorescens.Secondly,effects of different intensity levels of ultrasound on removing P.fluorescens biofilm formed on the surface of lettuce and the effects of ultrasonic cleaning on the quality of lettuce during storage were explored.High-intensity ultrasound showed a high efficacy of removing biofilm formed on the surface of lettuce.The intensity of ultrasound at 15.79 W/cm²only reduced 51.3%of the microorganisms on the surface of lettuce;the intensity of ultrasound at 26.32 W/cm² reduced 94.7%of the biofilm on the surface of lettuce and prolonged the storage period of lettuce upto 2 days.At the 4^th day,the total number of colonies on the surface of lettuce exceeded 10⁵CFU/g.In addition,the ultrasonic treatment slowed down the weight loss rate,chromatic aberration,and increased malondialdehyde formation in lettuce during cold storage.The ultrasonic treatment would also delay the decrease of p H,total phenolic content,and the degradation of chlorophyll in the lettuce.Lastly,the acoustic-thermal synergistic treatment on P.fluorescens biofilm was conducted to achieve the removal of the biofilm and the sterilization of the internal bacteria.Firstly,models of COMSOL multi-physics,i.e.frequency domain pressure acoustics,non-isothermal flow,K-εturbulence,and heat transfer model were coupled simultaneously.The ultrasonic pressure and temperature distribution in vessel and inner wall during the synergistic acoustic-thermal process were simulated.Results showed that sound pressure in the center of the biofilm reached1.67×10⁴,1.93×10⁴,and 2.15×10⁴ Pa when the ultrasonic input intensity was 15.79,21.06,and26.32 W/cm²,respectively.When the ultrasonic intensity was 26.32 W/cm²,the core temperature reached 43.96,53.89,and 64.53°C,respectively.With the increased distance between the target location and the ultrasonic probe,the temperature dropped rapidly to the temperature of the external circulating cooling water.The existence of high/low areas of sound pressure on the inner wall where the biofilm grows indicated potentially sanitary dead ends where the biofilm would not be effectively removed.Therefore,changing the sonication-heat treatment conditions or optimizing the design of container would avoid the dead ends and ensure the safety of foods caused by biofilm residues.Different conditions of ultrasonic-thermal treatment（US-TM）had significant impacts on biofilm removal and the sterilization of internal bacteria.The ultrasonic power of was higher than 21.06 W/cm²and the temperature was higher then 50°C would achieve an efficient P.fluorescens biofilm removal（>65.6%）and sterilization of internal bacteria（>99.9%）.Therefore,the US-TM would become a potential mean of achieving foodborne microbial biofilm control in the food industry.