| Pressurized CO2treatment was a food sterilization technology that using the temperature above or close the critical temperature of31.1℃and the pressure above or close the critical pressure7.13MPa. CO2was a natural and safe antiseptic. The sterilization temperature of pressurized CO2technology was low. CO2could be separated completely by decompression after sterilization. Pressurized CO2treatment was considered as a very promising cold sterilization. The purpose of this project is to investigate the bactericidal effects of pressurized CO2on the bacteria in saline, analyse the influencing factors and the residual bacterial activity; investigate the sterilization mechanism of pressurized CO2; compare the sterilization effects of pressurized CO2and thermal pasteurization on natural microorganisms in raw milk and the growth of natural microorganisms during storage; investigate the effects of pressurized CO2on protease and lipase activity in raw milk and the change of activity during storage. The application of pressurized CO2sterilization techniques to the dairy industry would be a technological innovation, the research of this subject would provide the theoretical basis for CO2cold sterilization technology, and the research of this subject had the significant meaning to the pressurized CO2sterilization of milk and dairy products.Escherichia coli, Pseudomonas fluorescens, Bacillus subtilis and Staphylococcus aurens in saline were treated by pressurized CO2. The factors influencing the bactericidal effects were studied by changing the pressure (1.5MPa,3MPa,4.5MPa,6MPa and7.5MPa), time (20min,40min,60min,80min and100min), temperature (30℃,35℃,40℃,45℃and50℃), media (saline, skim milk and whole milk) and modus (40min,20min×2and10min×4). The change of residual bacterial activity was also studied (30℃,7.5MPa, treated by pressurized CO220min,40min and60min). The results showed that bactericidal effects of pressurized CO2were increased when the pressure, time, temperature were increased. The milk ingredients could protect the bacteria. Changing the pressurized modus under low pressure could not increase the bactericidal effects. The bactericidal effects of pressurized CO2to the four bacteria were Pseudomonas fluorescens> Escherichia coli> Bacillus subtilis> Staphylococcus aureus. The bactericidal effects of pressurized CO2to Gram-negative bacteria were better than Gram-positive bacteria. Pressurized CO2had the subsequent damaging effects to residual Escherichia coli and Pseudomonas fluorescens, the growth lag phases of residual bacteria were extended a lot. But pressurized CO2did not have the subsequent damaging effects to residual Bacillus subtilis and Staphylococcus aureus, the growth lag phases of residual bacteria were not extended.Escherichia coli (7.5MPa,25℃/45℃,10min/20min/30min/40min/50min) and Bacillus subtilis (7.5MPa,25℃/45℃,20min/40min/60min/80min/100min) in saline were treated by pressurized CO2Compared with pasteurization (63℃,30min), the sterilization mechanism of pressurized CO2was studied by detecting the change of membrane permeability, the leakage of bacterial protein, nucleic acid, K+and Mg2+, the change of bacterial ultrastructure, the change of bacterial DNA. The change of bacterial components and bacterial protein secondary structure were also studied by FTIR. The results showed that the membrane permeability was increased (P<0.05) when the bacteria were inactivated by pressurized CO2, but differently from thermal pasteurization, the transmissive changes were not happened. Protein leakage was not happened under pressurized CO2treatment of short time. Although the protein leakage under pressurized CO2treatment of long time was more than (P<0.05) thermal pasteurization significantly, the time was lagged to the time of99%bacterial death. So protein leakage under pressurized CO2treatment of long time was not the reason of bacterial death, was only the secondary phenomenon of bacterial death. Pressurized CO2treatment induced the bacterial nucleic acid leaked significantly (P<0.05), so bacterial death was related to the bacterial nucleic acid leakage. Bacterial death was related to the bacterial K+and Mg2+leakage induced by pressurized CO2treatment. Pressurized CO2treatment destroyed the cell wall and membrane, bacterial deformation was happened. It was also certified that the bacterial membrane permeability was increased and the bacterial components were leaked. The degradation of bacterial DNA was not happened after pressurized CO2treatment, so bacterial death was not related to the damage of bacterial DNA. It was known through FTIR that pressurized CO2treatment induced the change of bacterial fatty acid and nucleic acid. The peptidoglycan layer of Escherichia coli changed obviously, the peptidoglycan layer of Bacillus subtilis did not change obviously. The protein of Escherichia coli changed obviously, the protein of Bacillus subtilis changed erratically. Differently from thermal pasteurization, pressurized CO2treatment induced the a-helix structure of Escherichia coli decreased, the a-helix structure changed to the β-sheet structure and the β-turn structure. The β-sheet structure of Bacillus subtilis increased, the change of a-helix structure, p-turn structure and the random coil structure of Bacillus subtilis were irregular.Raw milk was treated by pressurized CO2. The inactivation effects of pressurized CO? and pasteurization on natural microorganisms in raw milk and the stability of the treated raw milk during storage were investigated comparatively. The effects of pressurized CO2treatment parameters on the inactivation results were investigated by changing CO2pressure (4.5,7.5and10.5MPa), pressurized time (20,40and60min), temperature (4,15and25℃) and pressurized modus (continuous treatment and intermittent treatments). The effect of pressurized CO2treatment at the optimal conditions on the storage stability of natural microorganisms in raw milk at4℃was investigated and compared with the samples pasteurized at63℃for30min. The results showed that the viable counts of Pseudomonas, Enterobacteriaceae, LAB and Staphylococcus aureus in raw milk were decreased and the total bacterial count was also decreased. The CO2pressure, pressurized time and temperature had more profound effects on the inactivation degree while the effect of pressurized modus was less profound relatively. The gram-positive bacteria in raw milk were more resistant to the pressurized CO2treatment than gram-negative bacteria. At7.5MPa,4℃, under continuous pressurized CO2treatment for40min, the viable count of Staphylococcus aureus was decreased to the level undetectable, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count were decreased to the levels lower than pasteurization, and the viable count of LAB was more than pasteurization. During storage, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count in the samples treated by pressurized CO2were lower than which in the samples treated by pasteurization, the viable count of LAB was more, Staphylococcus aureus was undetectable. Compared with pasteurization, the storage time of raw milk was extended two days.Raw milk was treated by pressurized CO2(7.5MPa,25℃/45℃,20min/40min, continuous treatment). The results showed that compared with the protease activity of untreated raw milk3.61U/mL and the lipase activity of untreated raw milk0.43U/mL, when not degassed after pressurized CO2treatment, the protease activity (2.92U/mL,2.92U/mL,2.40U/mL,2.20U/mL) and lipase activity (0.25U/mL,0.26U/mL,0.16U/mL,0.12U/mL) of pressurized CO2treated raw milk were decreased significantly (P<0.05), when degassed after pressurized CO2treatment, the protease activity (3.30U/mL,3.42U/mL,3.24U/mL,3.21U/mL) of raw milk was increased slightly (P>0.05) and the lipase activity (0.55U/mL,0.51U/mL,0.57U/mL,0.52U/mL) was increased significantly (P<0.05). Pressurized CO2treatment could inhibit the proteolytic of raw milk during storage, and the inhibition effect was more when under short time, but the effect of temperature was not more. Pressurized CO2treatment induced the lipolysis of raw milk increased during storage, and the degree of lipolysis was more under long time and low temperature. The smaller fat globules induced by pressurized CO2treatment was the reason of the speeded-up of lipolysis. |
PDF Full Text Request