Identification, Creation, and Characterization of Bio-Based Antifreeze Agents

Ice recrystallization is a key problem leading to quality deterioration of frozen foods and biomedical materials. Molecules possessing ice recrystallization inhibition (IRI) activity can control ice crystal size after freezing, reducing the degree of freezing-induced damage. While these IRI active molecules have shown promise in maintaining the quality of frozen foods, their slow discovery, limited creation strategies, and lack of mechanistic understanding limit their hypothesis-driven design. In this work, we address these above-stated needs required for the streamlined implementation of these materials into frozen systems by the creation of a machine-learning-based IRI screening method, the development of a new IRI agent creation strategy, and through the study of a small molecule IRI agents’ mode of action. Cellpose, a segmentation algorithm, was used to develop an automated ice measurement protocol to better screen IRI agents using images from the splat and sucrose sandwiching assays. Next, Ni²⁺ immobilized metal affinity chromatography (IMAC) was used to fractionate pulse peptide hydrolysates by their histidine, cysteine, and tryptophan residues into peptide fractions enriched or lacking these amino acids. The fractionation of these hydrolysates induced IRI independent of their evaluated molecular characteristics, demonstrating for the first time that IMAC fractionation of pulse peptides may act as a viable creation strategy for bio-based IRI agents. Lastly, we present the discovery of a new IRI agent from biomass, sodium copper chlorophyllin (SCC), that exhibits potent IRI in 10 mM NaCl without inducing IRI in 180 mM NaCl and PBS. SCC’s anti-freeze activities only included IRI, lacking dynamic ice shaping and thermal hysteresis. SCC’s IRI was not mediated by ice binding, self-assembly, or governed by its surface orientation within 10 mM NaCl. Hence, this Thesis advances the field of cryopreservation by the novel identification, creation, and characterization of bio-based IRI agents.