The Impact Of Dense Phase Carbon Dioxide On Membrane Permeabilization And Proteins Of Escherichia Coli

Dense phase carbon dioxide is a new non-thermal sterilization technique for green food with several advantages including low treatment temperature, safety and pollution-free. However, few studies were reported on the sterilizing mechanism of dense phase carbon dioxide. In particular, the change of the membrane permeability and proteins inside the cells was rarely reported. In this paper, dense phase carbon dioxide effect on membrane permeability and the intracellular proteins of E. coli was studied, in order to explain the sterilizing mechanism of dense phase carbon dioxide.The influence of membrane permeability of E. coli treated by dense phase carbon dioxide was studied through determination of the content of the release of protein, nucleic acid, Mg²⁺, K⁺ and malondialdehyde (MDA) coupled with Transmission Electron Micrographs (TEM) technique in the experiment. Under the conditions of 7 MPa, 37℃for 10 min, 99% of E. coli was inactivated by dense phase carbon dioxide . Proteins, nucleic acid and Mg²⁺, K⁺ leaked from the cells, the content of MDA increased and the density of substances inside the cell decreased through the TEM view. The results indicated that it was an important cause of cell death that the membrane permeability of E. coli increased after E. coli was treated by dense phase carbon dioxide treatment.Proteomics analysis was used to analyze the change of proteins inside E. coli which was treated by dense phase carbon dioxide in this study. Comparison of two-dimensional electrophoresis protein profiles of E, coli treated and non-treated by dense phase carbon dioxide revealed that the abundance of 46 protein spots had 3-fold significant difference (p<0.05) in response to dense phase carbon dioxide treatment. Among them, the abundance of 16 protein spots was remarkably altered. MALDI-TOF MS was used to identify the 16 proteins. Of them, three proteins (spots 1, 4 and 13) were involved in cytoskeleton organization, nine proteins (spots 5, 6, 7, 8, 9, 10, 11, 12 and 15 ) were related to metabolism and four proteins (spots 2, 3, 14 and 16) were related to DNA. The results indicated that the change of some main proteins and enzymes inside the cells, which were caused by dense phase carbon dioxide treatment, may inhibit a series of normal physiological activities and lead the cells die.The impact of dense phase carbon dioxide on the structure of acidic proteins within different range of molecular weight inside the E. coli was studied by the method of circular dichroism combined with gel chromatography. The results showed the largest decline in the value ofα-helix of acidic proteins with the molecular weight range of 31000 20000 Dal treated by dense phase carbon dioxide, and the smallest decline with molecular weight range of less than 21000 Dal.α-helix had the trend of translating intoβ-Folded and random coil. The value ofβ-sheet of acidic proteins with the molecular weight range of 43000 31000 Dal had the largest raise. Differently, the value ofβ-turn of acidic proteins with the whole molecular weight range had no significant change. The results showed that dense phase carbon dioxide treatment resulted changes of acidic protein structures with different range of molecular weight, leading the loss of biological activity of the proteins. Therefore, it may impede the normal physiological activity of the cell and lead to cell death.