| In order to obtain a better understanding the electroporation behavior cell membrane,vesicles made by Soya PC,DPPC,DOPC encapsulated with 5(6)-CF were used as a cell membrane stimulation to evaluate the membrane electro-permeabilization induced by PEF.Effects of size,membrane lipid composition and membrane fluidity of vesicles on its electropermibiliztion were explored.Finally,the relationship between cell membrane fatty acid composition and PEF resistance of E.coli and L.monocytogenes were investigated.Results were as follow:Firstly,the particle and stability properties of phosphatides dispersions affected by PEF,US,MF treatment were compared.Results demonstrated that it was feasible that vesicles made by phospholipids were used as a stimulation of cell membrane to evaluate the cell membrane electro-permibiliztion under PEF treatment.Secondly,effects of PEF on electro-permeabilization of Soya PC vesicles with different size were investigated.Results indicated that the electro-permeabilization of LUV was increased with increasing intensity of electric field(> 10 kV/cm)and treatment time.However,for SUV,no siginificant changes were observed.Those results demonstrated that the magnitude of the induced transmembrane potential on the lipid membrane was proportional to the radius of the vesicle,and larger vesicles could be polarized or ruptured at relatively lower field strength than smaller vesicles.Thirdly,the permeability of Soya PC and DPPC vesicles under separated or combined thermal and PEF treatments was investigated,respectively.Results indicated synergetic effects of PEF and thermal treatment on electro-permeability of two vesicles were apparently different.For soya PC vesicles,the increment degree of R value was almost the same under various combination treatment conditions.However,for DPPC vesicles,it was less sensitive to PEF treatment.There results demonstrated that the electro-permeabilizaiton cell membrane was directly related to the membrane fluidity and its molecular structure change during phase transition.And then,the effects of vesicle components on its electro-permeability were investigated.Results demonstrated that the membrane electro-permeability was directly related to the membrane composition vesicles.Compared to the DPPC vesicles,DOPC vesicles were much more sensitive to PEF treatment.In addition,the electro-permeability of DOPC vesicles decreased sharply with the increase of DPPC molar ratio.For the soya PC vesicles,the R was dropped sharply from 48.13 % to 24.11 % with the molar ratio of cholesterol increased from 0-50 %.Raman spectroscopy analysis revealed that the electro-permeability decrease of vesicles with the addition of cholesterol was responsible to the cholesterol incorporated into hydrocarbon partition of lipid bilayers,leading to the order arrangement of acyl chain of phospholipid,which resulted in the increase of bilayer membrane thickness and the decrease of fluidity of bilayer membrane.Finally,the underlying relationships between m FAC and PEF resistance of E.coli and L.monocytogenes were explored.Results indicated the PEF resistance of E.coli and L.monocytogenes at stationary stage was directly related to its mFAC.For the E.coli cells at the stationary stage,as growth temperature decreased from 37 °C to 15 °C,the PEF(23.3 kV/cm,1.2 ms)lethality of E.coli was increased 1.5 log10,reached 3.2 log10.Meanwhile,the proportion of unsaturated fatty acid(UFA)was increased from 20.07 to 47.65 %.And the fluidity index(FI)of the cell membrane was also increased from 0.42 to 1.42.It revealed that the PEF resistance of E.coli at stationary stage was determined by the mFAC and fluidity of the cell membrane.Those conclusions were confirmed by cytomembrane fluidity determined by fluorescence probe(DPH)and the different electro-permeabilization DOPC and DPPC vesicles.For L.monocytogenes,Results were the same as the studies about E.coli,excepting the PEF resistance of L.monocytogenes was directly related to the content of branch chain of fatty acid in cell membrane.The fraction of anteiso-C15:0 in cell membrane decreased with increasing growth temperature in both exponential and stationary stage cells which correlated closely with the fluidity index(FI)of cell membrane.In stationary stage cells,the PEF resistance directly related to the fluidity of cell membrane.The greater fluidity of cell membrane was,the lower PEF resistance was.However,for the cell at exponential stage,there was apparently no obvious relationship between membrane fluidity and PEF resistance,indicating that the fluidity of cell membrane is not the dominant factor determining the PEF resistance of cells at exponential stage. |
