141 Inhibition of ice recrystallization by antifreeze proteins

Abstract

Ice recrystallization (IR) is a process in which large ice crystals grow at the expense of smaller ones. It occurs constantly in nature and in industrial processes in conditions of moderate cooling of a partially frozen environment or at accelerated rate due to temperature fluctuations of frozen substances. IR is one of the major causes of smooth texture loss and deterioration of frozen food such as ice cream during storage. In cryopreservation, recrystallization during thawing, damages the cells and tissues due to membrane rapture and cell dehydration, reducing the survival rates. Organisms inhabiting cold environments and prone to IR injuries, such as freeze-tolerating plants and freeze-avoiding organisms like fish and insects have developed antifreeze proteins (AFPs), a subset of the Ice Binding Proteins group which interact with ice for various biological purposes. The AFPs adsorb to ice surfaces and restrict the growth of ice to the areas between bound protein molecules. Such proteins were shown to block IR effectively [Knight and Duman, 1986], and may serve as additives in many applications in which ice recrystallization is an obstacle. Although ice recrystallization inhibition (IRI) activity of a variety of AFPs was studied [Knight et al., 1995], the mechanism by which AFPs inhibit IR is unknown. Particularly, it has been shown that although insect AFPs have considerably higher freezing point depression activity relative to AFPs from fish and plants, their IRI activity is in the same range or lower [Yu et al., 2010]. We intend to study the mechanism of IRI in order to explain the inconsistency between this activity and the freezing point depression activity of insect AFPs. In our study the IRI activity of AFPs from mealworm Tenebriomolitor (TmAFP) was investigated. Sucrose solutions containing various concentration of TmAFP were cooled to −50 °C, then the temperature was elevated and sustained constant for annealing. During annealing, images of recrystallization were acquired using a light microscope equipped with fluorescence illumination and cold-stage. Initial results show an accumulation of TmAFP onto ice crystals and IRI in a range of AFPs concentrations. Our next goals are to develop test methods that will provide reliable, quantitative information on recrystallization and on the accumulation of the protein on the crystals, as well as depletion of the protein from the solution. Such methods will enable quantification of the ice recrystallization phenomena and subsequently quantification of the differences between the IRI activities of various types of AFPs. Understanding the mechanism of ice recrystallization and the effects of AFPs on it may improve and expand the use of AFPs in many applications in the medical sector, in cryopreservation, and in the frozen food industry. Source of funding: European Research Council (ERC). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il