| Extracellular ice nucleators (ECINs) are membrane vesicles released by E. herbicola, which can initiate ice nucleation at {dollar}{lcub}-{rcub}2spcirc{dollar}C. The objectives of this study were to develop an accurate method for quantitative evaluation of ice nucleation activity (INA), to enhance the production of ECINs from E. herbicola, to isolate and characterize ECINs, and to investigate the INA of purified ECINs in the freezing of selected foods.; This work demonstrated that when the frozen fraction (f) is less than 40%, the probability of ice nucleation in a population of droplets follows a Poisson distribution. Therefore, it was established that valid values of f must be 40% or less in Vali's equation for an accurate assay of INA.; Optimal production of ECINs was achieved when cells were grown in yeast extract to early stationary phase at temperatures below 22{dollar}spcirc{dollar}C. ECIN production was enhanced by 5-fold when cells were treated with EDTA and by 20- to 30-fold when subjected to sonication, a yield of 840 mg ECIN protein per liter of cells.; ECINs were further isolated using ammonium sulfate precipitation, sucrose density gradient centrifugation and gel filtration chromatography with a 27-fold increase in specific activity. The INA of ECINs varied with pH and the presence of Mg{dollar}sp{lcub}2+{rcub}.{dollar} INA was eliminated by organic solvents, detergents or proteolytic treatments. Lipases, {dollar}alpha{dollar}-mannosidase and {dollar}beta{dollar}-galactosidase also inactivated the INA of ECINs. It was established that ECINs are particulate in nature and their ability to facilitate INA depends upon the physical and chemical integrity of membrane vesicles. These vesicles can become larger in size via aggregation and/or fusion.; Similar to the application of whole cells, addition of purified ECINs to liquid foods such as milk and juices significantly elevated their ice nucleation temperatures and promoted freezing. Freezing of solid or semi-solid products such as tofu and yogurt in the presence of ECINs resulted in fiber-like textures, which were unique relative to controls. The results with the differential scanning calorimetry (DSC) revealed that ECINs initiate phase transition at a higher temperature but extend the whole time period for the phase transition relative the control, by which ECINs change ice-formation patterns and consequently lead to unique texture formation. One of the advantages of using ECINs, as shown in this work, is that a low concentration level relative to whole cells (compare 1 {dollar}mu{dollar}g protein per ml to 10{dollar}sp7{dollar} cfu per ml) is required to obtain the same effect on freezing. |
