Abstract:

【Objective】Effects of heat stress on cell membrane and membrane protein of Escherichia coli were investigated with ATCC43889 as the test microorganism in this work, which would provide a theoretical basis for the application of high temperature sterilization technology in the food industry. 【Method】 The changes of the cell membrane and membrane protein for three heat-resistant ATCC43889 strains which were treated with 10 times of heat stress treatments at 50℃, 60℃, and 70℃ for 15 min, transferred into TSB and incubated again were studied, respectively. The morphologic changes of original control strain and the three heat-resistant strains of ATCC43889 were observed using scanning electron microscopy. The biofilm-forming ability for each strain was determined using a 96-well microplate method. The changes and differences of cell membrane fatty acid composition for each strain were monitored using gas chromatography. The changes of phase transition temperature of cell membrane phospholipid for each strain were measured using differential scanning calorimetry (DSC). The changes of outer membrane protein expression for each strain were examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 The experimental results showed that the individual morphology of ATCC43889 changed evidently after 10 times of heat stress treatments at 50℃, 60℃, and 70℃, transfers and incubation again, respectively. The individual morphology for a part of the cells changed from spheroids to long rods after heat stress treatment at 50℃. The individual morphology of the cells upon heat stress treatment at 60℃ was thinner and longer than that of the cells upon heat stress treatment at 50℃. Upon heat stress treatment at 70℃, most of the cells became longer rods, a large number of cells gathered together, and the surface of the cells was irregular and uneven. The vitality and ability of biofilm-forming for the three heat-resistant ATCC43889 strains enhanced with increasing heat stress temperature. The ability of biofilm-forming for the three ATCC43889 heat-resistant strains significantly (P<0.05) differ from that of the control group, while the ability of biofilm-forming for the heat-resistant strain at 60℃ was not significantly (P>0.05) different from that of the heat-resistant strain at 70℃. Among the fatty acids identified, three fatty acids were absent after 10 times of heat stress treatments at 50℃, 60℃ and 70℃ compared to the original control strain, which were C18:1n9c, C18:3n3, and C21:0, respectively. The amount of saturated fatty acid such as C13:0, C16:0, C17:0 and their total amounts increased, while the amount of unsaturated fatty acid such as C14:1, C16:1, C17:1, C18:1n9t, C18:2n6t and their total amounts in the cell membrane for the three heat-resistant strains decreased with the increase of heat stress temperature. The ratio of saturated to unsaturated fatty acids (SFA/USFA), the melting point (T_m) and the phase transition temperature of cell membrane phospholipids increased as the heat stress temperature increased. These changes in membrane fatty acid composition and the phase transition temperature of cell membrane phospholipids resulted in decreased membrane fluidity of the three heat-resistant strains. The protein band color of molecular weight about 63 and 75 kD deepened with the increase of heat stress temperature. The increase of heat-resistance for the two heat-resistant strains after 10 times of heat stress treatments at 60℃ and 70℃ was accompanied by synthesis of specific outer membrane proteins of molecular weight about 48 to 75 kD. These results indicated that synthesis and increased amounts of some specific outer membrane proteins could induce heat-resistance for E. coli ATCC43889. 【Conclusion】The longer individual morphology, the enhanced ability of biofilm-forming, the increased ratio of saturated to unsaturated fatty acids, the increased phase transition temperature of cell membrane phospholipid and the increased expression of some outer membrane proteins correlated with the greater heat-resistance of E. coli ATCC43889. These changes contributed to the adaptation of ATCC43889 to heat stress environment and improvement of cellular survival viability.

Key words: heat stress, Escherichia coli, biofilm, membrane fatty acid, SDS-PAGE