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The use of CO(2) under pressure (dense CO(2)) is one of the most promising techniques to achieve cold pasteurization and/or sterilization of liquid and solid materials, and is likely to replace or partially substitute currently and widely applied thermal processes. Although the ability of CO(2) to inactivate microorganisms has been known since the 1950's, only within the last 15 years it has received special attention, and the scientific and economic interest towards practical applications is presently growing more and more. Here we collect and discuss the relevant current knowledge about the potentials of dense CO(2) as a non-thermal technology in the field of microbial inactivation. We summarize the state of the art, including definitions, description of the equipment, relevant applications, in both simple suspensions and complex media, for the treatment of a wide range of microorganisms in both liquid and solid substrates. Finally, we also summarize and discuss the different hypotheses about the mechanisms of inactivation.

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We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive β-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.

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