So-called ‘anti-freeze proteins’ (AFPs) have the property of modifying the growth of ice crystals. They differ in their action from simpler ionic anti-freeze agents such as common salt. For example, aqueous AFP solutions typically have a freezing point that is lower than their melting point (hysteresis). They stabilise ice crystals over a range of temperatures, and inhibit re-crystallisation. They seem to assist organisms to survive in temperatures around the freezing point of water, and are accordingly found in several different types of organism. Their properties give them a range of potential uses: in particular in foods that are eaten while frozen, by inhibiting recrystallisation and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallisation during freezing, storage, transport, and thawing, thus preserving food texture by reducing cellular damage and also minimising the loss of nutrients by reducing drip (see Griffith, M. and Vanya Ewart, K. Biotechnology Advances, 13, pp 375-402).
Various sources of AFPs are known: the commonest are fish and plants. Some bacteria exhibit anti-freeze properties (see Griffith et al, supra, p 382). In a few cases, the isolation of anti-freeze proteins from bacteria has been reported (see for example: Xu H, Griffith M, Patten CL, et al., Can J Microbiol 44: (1) 64-73 January 1998). Given the advantages to an organism of resistance to freezing, it may be considered surprising that anti-freeze proteins are not more widely distributed in Nature and easier to find. This may be because organisms have other ways of overcoming such problems.
Whereas, fish AFP that can assist production of strong, hard structures has advantages for some specialised applications, an AFP that is RI-active but does not produce hard structures has its own, different advantages. For example, molecules that actively inhibit the recrystallisation of ice upon storage may be used to maintain smooth textures of frozen products during storage. For some frozen foods, such as dairy ice cream, it would be preferable if the use of such a molecule did not harden the product excessively.
Therefore there is a desire for anti-freeze peptides which are active in recrystallisation inhibition during thawing and freezing cycles, and which can be used for maintaining smooth textures of food products during storage. Desirably these anti-freeze peptides are heat stable such that they can be incorporated in food products which are pasteurized or sterilised, while they maintain their functionality.
The hypothesis on which the present invention is (in part) based is that bacteria are more likely to evolve AFP proteins if they inhabit a liquid aqueous environment which is often below the normal freezing point of water: in such environments (it is postulated) AFPs may be an efficient way of giving bacteria a competitive advantage.