Plant-inspired solutions for modern food technology: Ice Structuring Proteins from winter wheat

In a quest to find novel methods to improve food preservation, food companies product pipelines are largely driven by the growing customers’ request for natural solutions. To date, freezing is still the most efficient method to preserve perishable foods, without altering substantially its taste and texture and avoid the use of preservatives. The uncontrolled growth of ice crystals, pose however a serious risk for the stability of many frozen goods, threatening their integrity and sensorial features. A paradigm is represented by ice cream, whose ice crystals microstructure is fundamental to impart its characteristic mouthfeel, and as such, need to be preserved. A step forward toward the improvement of techniques to prevent extensive ice crystals growth, was represent by the introduction, a few years ago, of AntiFreeze proteins also known as Ice Structuring Proteins (ISP). By directly interacting with the water molecules that make up the crystalline structure of ice, ISP disrupt the regularity of those planes and inhibit, to a certain extent, further addition of water molecules and consequent ice crystal growth. To date, the only case of application of ISP in commercial products, is that of a fish ISP produced by recombinant S. cerevisiae. Besides the technological advantages in ice cream manufacturing enabled by the use of this protein, being a GMO derived product hampered its diffusion in GMO-skeptical communities, like in Europe and other western countries. In this scenario, the aim of this doctoral project, in collaboration with the University of Padova with the support of Food Research & Innovation srl, was focused on finding novel ISP alternatives for preservation of frozen foods. The discovery of novel sources and methods to obtain ISP, were driven by two requirement that needed to be satisfied: 1) the ISP source had be perceived as “safe” from potential consumers, 2) the bioprocess to obtain those ISP compatible with industrial uses. On the basis of requirement n.1, we chose wheat as our preferred source of ISP and, after a thorough literature search about wheat ISP, we decided to start our investigation from a Triticum aestivum Thaumatin-Like Protein (TaTLP), for which the gene sequence was publicly available. The choice fell on TaTLP as this protein was previously recovered from wheat extracts, with the ability to inhibit ice recrystallization in ice cream. In contrast with data reported in literature for TaTLP isolated from the plant, the recombinant product didn’t show any ability to prevent ice crystals grow. After a thorough biochemical investigation, we concluded that the lack of activity wasn’t ascribable to incorrect processing or maturation of TaTLP, whose structural features resembled much that of other proteins belonging to the same family. Our hypothesis, is that TaTLP doesn’t exhibit any activity toward ice per se and that previous studies suggesting the opposite, may have derived from isolations of homologues TLP, that evolved ice binding domains. From our investigations on a wheat apoplastic extract containing ISPs, we found that the activity of those ISPs could be enhanced upon addition to the extract of exogenous proteins, not necessarily ISPs. At the moment, we don’t have strong experimental evidences to elucidate the mechanism at the molecular level, but we believe that some wheat ISPs, may have evolved a strategy to enhance their single contribution to the total ice growth inhibition activity of the plant. This enhancement may rely upon the interaction of wheat ISPs with other proteins, similarly to what was observed for AFP from the overwintering insect D. canadensis. In the search of a method to efficiently extract ISP from wheat that could be used as stabilizers of the ice phase in ice cream, we set up a one-step extraction process that involved high temperature treatment of the plant. This method represent a significant step forward in terms of simplicity and yield of ISPs, compared to previous extraction methods. Plus, the ISPs obtained thereof are heat resistant, an essential feature for proteins which are to be used in industrial foods. Wheat ISPs were then used to formulate ice cream in pilot pre-industrial plant; these ice cream were subjected to thermal shock to exacerbate temperature fluctuations experienced by ice cream in its path from the factory to the household freezers. The result is a stabilized ice cream, in which ice crystals growth was inhibited by the addition of a wheat extract containing ISPs. This extract could be considered as a natural ingredient, lacking the need to be declared as an additive and therefore not falling under the E-number labeling regulation for food ingredients. In parallel to the tests performed on ice cream, isolation and partial characterization of ISP active species from wheat extract were attempted. At the end of the purification process, comprising 3 consecutive chromatographic separations, 5 proteins were eluted in a single fraction showing activity towards ice crystals growth. Being that these proteins have very similar isoelectric points and electrophoretic mobility, we believe that they might be isoforms of the same species. To conclude, we designed a feasible process to obtain ISP from a vegetal source that successfully stabilized ice cream, this method having all the features required to be implemented on a larger scale for industrial production purposes. From the collateral results obtained from our investigations on wheat extracts, interesting clues about the nature of wheat ISP has emerged, as well and their possible roles in preventing ice growth in a physiological context. Those findings, contribute to broaden the knowledge of a category of ISP that is still poorly characterized and pose the basis for new research lines.