Research On Inactivation Effect Of Pulsed Light On Escherichia Coli And Its Mechanisms

In order to pursuing high-quality life,people are increasingly focusing on the issues of food quality and safety and pay more attention to food sterilization technologies.Pulsed light(PL)as a novel sterilization technology has become one of the research hotspots in the field of food sterilization because of its high sterilization efficiency,flexible operation,and no pollution to the environment.However,the current studies on PL sterilization mainly focus on the sterilization effect and application.There are few researches on its sterilization mechanism,especially the photothermal sterilization mechanism.Escherichia coli(E.coli)was used as object microorganism to study the inactivation caused by PL and its mechanisms of photothermal sterilization.Inactivation effect and kinetic model of pulsed light treatment on E.coli were studied.Morphological structure,cell membrane fluidity and permeability as well as proteomics of E.coli were also discussed.Photothermal sterilization mechanisms were explored preliminarily from the cellular level and the molecular level.The main research work and its results were as follows:(1)Different growth periods of E.coli and physical properties(temperature,pH and transmittance)of suspension have a great effect on PL inactivation.E.coli cultured for 6 h at logarithmic phase was most sensitive to PL and the treatment of 1 s could reduce2.31±0.16 lg cfu/mL.High temperature(35~45?),acidic(p H4.0~6.0)or alkaline(pH8.0)conditions could promote the inactivation influence of PL.What's more,the lower the light transmittance of the suspension was,the worse the bactericidal effect was.When E.coli was cultured for 6 h,the temperature of the suspension was 35°C,pH was 4.0,light transmittance was 22%,pulse distance was 13 cm(0.35 J/cm~2/pulse)and pulse frequency was 4 pulses/s,E.coli treated for 1 s could be reduced by 6.27±0.02 lg cfu/mL.(2)PL equipment parameters like pulse distance and pulse time also affect the bactericidal effect significantly.Under the two different PL equipment,both Linear model and Weibull model could fit the pulse light sterilization kinetic curve(R~2>0.99)well,and the Weibull model was more accurate than the Linear model.In addition,it can be found that the inactivation caused by pulsed light depended on the way of fluence dosage according to the kinetic curve with the fluence as a variable.While it was irrelevant whether the fluence was regulated by the time or the distance.(3)The scanning electron microscopy(SEM)observation indicated that PL caused deformation on the surface of E.coli with atrophy,wrinkling and perforation,disrupting cell wall and cell membrane structure.(4)Fluorescence polarization technique was used to determine the membrane fluidity by labelling the E.coli at 55?for 40 min with the fluorescence probe DPH.The results showed that PL could improve the fluorescence polarization and fluorescence anisotropy,meaning that membrane fluidity decreased.In addition,as the pulse time increased,more propidium iodide dyes entered into the cell membrane,accompanied by leakage of electrolytes and intracellular macromolecules like nucleic acids and proteins,indicating cell membrane permeability increased.Changes in the fluidity and permeability of the cell membrane would result in the lost of the membrane function,which may cause the death of E.coli.(5)Proteomic method based on isobaric tags for relative and absolute quantitation(iTRAQ)was used for compare the protein profile of E.coli PL-treated(low-dose group:48s with 100 J;high-dose group:96 s with 100 J)and PL-untreated.25 and 249 differential proteins were obtained in the low dose vs the blank group and the high dose vs the blank group respectively by differentially screening.GO and KEGG pathway analysis were employed to analyzed the differential proteins.Results indicated that PL continued to destroy the physiological functions of E.coli with the increase of radiation dose,eventually causing damage to the cell membrane and intracellular substances.At the same time,PL affected organics and energy metabolism of E.coli,especially for pathways of aminobenzoate degradation,ABC transporters,flagellar assembly and bacterial chemotaxis,which may cause the cell death.