Research On Inactivation Of Food Bacteria By The Combined Treatment Of Carbon Dioxide With High Hydrostatic Pressure

High hydrostatic pressure (HHP) can be used to inactivate microorganisms while allows a better retention of product flavor, texture, color, and nutrient content than a comparable conventional heat pasteurization, avoiding heat destroying on these thermally sensitive ingredients. However, the expensive cost for the construction and running of HHP device is caused by very high pressure required for the inactivation of pressure resistant pathogenic and spoilage bacteria. This has limited the commercial breakthrough of HHP technology. Recently, to reduce the inactivation pressure, various effective synergistic treatments have attracted much more attention. These combined factors include antimicrobials, pH, moderate temperature and cycled pulse pressure. For the antimicrobials from natural sources, such as nisin, pediocin, lysozyme, lactoperoxidase and CO2, have been used to combine with HHP for microbial inactivation. It has been found that several natural biopreservatives, such as lysozyme and bacteriocins, are effective against some gram-positive bacteria by acting on the cell wall. Due to the protection from the outer membrane against the penetration of the above peptides and enzymes, gram-negative bacteria are normally insensitive to these antimicrobials. What is more, the addition of biopreservatives goes against "zero-added" principle. The combination of moderate heat also caused damage of nutrient and flavor to heat sensitive juices. Carbon dioxide is a natural inhibitory compound for microorganisms. It is cheap and safe for use. The combination of CO2with HHP can increase inactivation, thus decrease the treatment pressure. However, the low and unstable CO2concentration in the use with HHP has limited the inactivation effect. This limited the investigation and application of the combined method. Furthermore, the mechanisms of the synergistic bactericidal action on microorganisms remained unclear, which included the action of HHP, the action of CO2under pressure and the synergistic action of both on bacteria cells.In this dissertation, a novel experimental method for the combined treatment of CO2and HHP was designed. A device was constructed to carry out the inactivation process at given CO2concentrations. The inactivation of Staphylococcus aureus and E. coli by the combined treatment, as well as the influence of CO2concentrations, treatment pressure and temperature on the bactericidal effect, were studied. The mechanisms of the synergy of CO2and HHP, including pressure induced CO2permeation of the cell membrane and denaturation of intracellular protein, were investigated. The combined treatment was applied in the pasteurization of the fruit juice to inactivate the common pathogenic and spoilage bacteria. The influence of fruit juice pH and matrix on the outcome of the combined treatment was studied to optimize the treatment parameters and establish the basis for the development and application of HHP technology. The main research results obtained as follows:1. With the addition of3～5.5NL/L CO2in the bacteria suspension, the Staphylococcus aureus and Escherichia coli can be inactivated at pressure200-250MPa lower than that without CO2.The effective treatment condition was at3.8NL/L CO2,350MPa and30℃for10min for S. aureus and3.2NL/L CO2,250MPa and30℃for10min for E.coli. The bactericidal pressures were reduced by200-250MPa compared to HHP alone. The inactivation rate increased rapidly with the increasing of the CO2concentration and leveled off, reaching the maximum inactivation at around5NL/L CO2. Temperature affected the inactivation significantly. The inactivation was effective at30℃or higher than30℃and declined clearly at below20℃. Higher or cycled pressure could be applied to acquire effective inactivation at lower temperature. Considering HHP as a true cold pasteurization method, the optional treatment temperature could be at25-35℃. While CO2in combination with HHP increased the inactivation, it increased the sublethal injury on bacteria cells. The combination of CO2also increased dynamic pressure inactivation to some extent. However, different from HHP treatment, hig pressure homogenization (HPH) did not caused sublethal injury on cells.2. CO2destroyed the integrity of bacterial cell membrane, lowered the intracellular pH and facilitated protein aggregation under high pressure. These accounted for its bactericidal synergy with HHP.The scanning electron microscope (SEM) images of the E. coli and S. aureus showed a highly deformed morphology of a rough surface with many concave defects and ruptured cell wall, whereas the cells treated with pressure alone only showed invaginations with the surface remaining smooth and continuous. The damage of E. coli cell wall was more serious than S. aureus. The results indicated that the combination of CO2increased the damage of bacteria cells.Laser scanning cofocal microscope (LSCM) image and flow cytometry (FCM) analysis revealed that the combined treatment caused much more permeability of cell than HHP alone. The percentage of PI stained E. coli cells increased from12%to87%and S. aureus cells from5%to69%. CO2increased membrane permeability which was one of the key mechanisms involved in the inactivation of cells.The transmission electron microscope (TEM) image of ultrathin section of E. coli cells showed that CO2in combination with HHP induced the intracellular protein aggregation. CO2permeation resulted in the decrease in intracellular pHi. The cytoplasmic protein with high concentration, including ribosome aggregated under high pressure, destroying the reactive compounds and inactivating cells. The intracellular protein aggregation was another key mechanism involved in the inactivation by the combined treatment.The inhibition test of CO2permeation indicated that5mM PBS inhibited permeation of0.2M CO2entirely. A model of CO2permeation of the cell membrane was erected as follows:when a bicarbonate absorbed on the membrane phosphatide combined a proton, a CO2was created and permeated into the cell. When a dihydrogen phosphate ion displaced the bicarbonate on the membrane, CO2permeation was inhibited and its synergy on HHP inactivation was inhibited.3. The selected vegetative bacteria E. coli, L. innocua and L. plantarum were effectively inactivated by the combination of dissolved4.5NL/L CO2and mild pressure350MPa or below in orange, tomato and carrot juices. Moreover, the synergy of CO2increased the sublethal damage and further inactivation during storage.The present study used four fruit juices, orange (pH3.4and pH3.8), tomato (pH4.2) and carrot juice (pH6.3) in HHP treatment with or without dissolved CO2. The results indicated that the product matrix, such as soluble solids and suspended particles, did not exhibit an apparent influence on the action of the CO2. The juice pHs displayed different influences on the inactivation depending on pH<4or pH>4. The inactivation in tomato and carrot juices at pH4.2and6.3showed minor difference with that in physiological saline. The juice pH exerted no significant influence (p<0.05) on the inactivation at pH higher than4. By comparison, the combined effect was considerably promoted in orange juice at pH3.8, while the HHP inactivation was enhanced to a limited extent. In another orange juice of pH3.4, all the three strains lost their pressure resistance. HHP alone completely inactivated E. coli at relatively mild pressures of200MPa, and L. innocua and L. plantarum at300MPa.Sublethal damage induced by the combined treatment at very low pressure increased the sensitivity of the bacteria to the storage pH and caused further inactivation in tomato juice during storage, whereas, low acidic carrot juice did not induce sufficient further inactivation, indicating that intensive treatment is required to ensure safety.