Design of eco-friendly antifreeze peptides as novel inhibitors of gas-hydration kinetics.

In this study, peptides designed using fragments of an antifreeze protein (AFP) from the freeze-tolerant insect Tenebrio molitor, TmAFP, were evaluated as inhibitors of clathrate hydrate formation. It was found that these peptides exhibit inhibitory effects by both direct and indirect mechanisms. The direct mechanism involves the displacement of methane molecules by hydrophobic methyl groups from threonine residues, preventing their diffusion to the hydrate surface. The indirect mechanism is characterized by the formation of cylindrical gas bubbles, the morphology of which reduces the pressure difference at the bubble interface, thereby slowing methane transport. The transfer of methane to the hydrate interface is primarily dominated by gas bubbles in the presence of antifreeze peptides. Spherical bubbles facilitate methane migration and potentially accelerate hydrate formation; conversely, the promotion of a cylindrical bubble morphology by two of the designed systems was found to mitigate this effect, leading to slower methane transport and reduced hydrate growth. These findings provide valuable guidance for the design of effective peptide-based inhibitors of natural-gas hydrate formation with potential applications in the energy and environmental sectors.

On the engulfment of antifreeze proteins by ice.

Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, [Formula: see text], for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, [Formula: see text], between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, [Formula: see text] scales as [Formula: see text] and not as [Formula: see text]. We further show that [Formula: see text] is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure-function relationships, we hope that our findings will pave the way for the rational design of AFPs.

A stacking-based algorithm for antifreeze protein identification using combined physicochemical, pseudo amino acid composition, and reduction property features.

Antifreeze proteins have wide applications in the medical and food industries. In this study, we propose a stacking-based classifier that can effectively identify antifreeze proteins. Initially, feature extraction was performed in three aspects: reduction properties, scalable pseudo amino acid composition, and physicochemical properties. A hybrid feature set comprised of the combined information from these three categories was obtained. Subsequently, we trained the training set based on LightGBM, XGBoost, and RandomForest algorithms, and the training outcomes were passed to the Logistic algorithm for matching, thereby establishing a stacking algorithm. The proposed algorithm was tested on the test set and an independent validation set. Experimental data indicates that the algorithm achieved a recognition accuracy of 98.3 %, and an accuracy of 98.5 % on the validation set. Lastly, we analyzed the reasons why numerical features achieved high recognition capabilities from multiple aspects. Data dimensionality reduction and the analysis from two-dimensional and three-dimensional views revealed separability between positive and negative samples, and the protein three-dimensional structure further demonstrated significant differences in related features between the two samples. Analysis of the classifier revealed that Hr*Hr, HrHr, and Sc-PseAAC_1, 188D(152,116,57,183) were among the seven most important numerical features affecting algorithm recognition. For Hr*Hr and HrHr, supportive sequence level evidence for the reduction dictionary was found in terms of conservation area analysis, multiple sequence alignment, and amino acid conservative substitution. Moreover, the importance of the reduction dictionary was recognized through a comparative analysis of importance before and after the reduction, realizing the effectiveness of the dictionary in improving feature importance. A decision tree model has been utilized to discern the distinctions between dipeptides associated with the physical and chemical properties of His(H), Iso(I), Leu(L), and Lys(K) and other dipeptides. We finally analyzed the other seven features of importance, and data analysis confirmed that hydrophobicity, secondary structure, charge properties, van der Waals forces, and solvent accessibility are also factors affecting the antifreeze capability of proteins.

Glycosylated Polyhydroxyproline Is a Potent Antifreeze Molecule.

Molecules that inhibit the growth of ice crystals are highly desirable for applications in building materials, foods, and agriculture. Antifreezes are particularly essential in biomedicine for tissue banking, yet molecules currently in use have known toxic effects. Antifreeze glycoproteins have evolved naturally in polar fish species living in subzero climates, but practical issues with collection and purification have limited their commercial use. Here, we present a synthetic strategy using polymerization of amino acid N-carboxyanhydrides to produce polypeptide mimics of these potent natural antifreeze proteins. We investigated a set of mimics with varied structural properties and identified a glycopolypeptide with potent ice recrystallization inhibition properties. We optimized for molecular weight, characterized their conformations, and verified their cytocompatibility in a human cell line. Overall, we present a material that will have broad applications as a biocompatible antifreeze.

Freezing and thawing in Antarctica: characterization of antifreeze protein (AFP) producing microorganisms isolated from King George Island, Antarctica.

Antarctic temperature variations and long periods of freezing shaped the evolution of microorganisms with unique survival mechanisms. These resilient organisms exhibit several adaptations for life in extreme cold. In such ecosystems, microorganisms endure the absence of liquid water and exhibit resistance to freezing by producing water-binding molecules such as antifreeze proteins (AFP). AFPs modify the ice structure, lower the freezing point, and inhibit recrystallization. The objective of this study was to select and identify microorganisms isolated from different Antarctic ecosystems based on their resistance to temperatures below 0 °C. Furthermore, the study sought to characterize these microorganisms regarding their potential antifreeze adaptive mechanisms. Samples of soil, moss, permafrost, and marine sediment were collected on King George Island, located in the South Shetland archipelago, Antarctica. Bacteria and yeasts were isolated and subjected to freezing-resistance and ice recrystallization inhibition (IR) tests. A total of 215 microorganisms were isolated, out of which 118 were molecularly identified through molecular analysis using the 16S rRNA and ITS regions. Furthermore, our study identified 24 freezing-resistant isolates, including two yeasts and 22 bacteria. A total of 131 protein extracts were subjected to the IR test, revealing 14 isolates positive for AFP production. Finally, four isolates showed both freeze-resistance and IR activity (Arthrobacter sp. BGS04, Pseudomonas sp. BGS05, Cryobacterium sp. P64, and Acinetobacter sp. M1_25C). This study emphasizes the diversity of Antarctic microorganisms with the ability to tolerate freezing conditions. These microorganisms warrant further investigation to conduct a comprehensive analysis of their antifreeze capabilities, with the goal of exploring their potential for future biotechnological applications.

The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Extended Temperature Range of the Ice-Binding Protein Activity.

Ice-binding proteins (IBPs) are expressed in various organisms for several functions, such as protecting them from freezing and freeze injuries. Via adsorption on ice surfaces, IBPs depress ice growth and recrystallization and affect nucleation and ice shaping. IBPs have shown promise in mitigating ice growth under moderate supercooling conditions, but their functionality under cryogenic conditions has been less explored. In this study, we investigate the impact of two types of antifreeze proteins (AFPs): type III AFP from fish and a hyperactive AFP from an insect, the Tenebrio molitor AFP, in vitrified dimethylsulfoxide (DMSO) solutions. We report that these AFPs depress devitrification at -80 °C. Furthermore, in cases where devitrification does occur, AFPs depress ice recrystallization during the warming stage. The data directly demonstrate that AFPs are active at temperatures below the regime of homogeneous nucleation. This research paves the way for exploring AFPs as potential enhancers of cryopreservation techniques, minimizing ice-growth-related damage, and promoting advancements in this vital field.

Antifreeze Polysaccharides from Wheat Bran: The Structural Characterization and Antifreeze Mechanism.

Exploring a novel natural cryoprotectant and understanding its antifreeze mechanism allows the rational design of future sustainable antifreeze analogues. In this study, various antifreeze polysaccharides were isolated from wheat bran, and the antifreeze activity was comparatively studied in relation to the molecular structure. The antifreeze mechanism was further revealed based on the interactions of polysaccharides and water molecules through dynamic simulation analysis. The antifreeze polysaccharides showed distinct ice recrystallization inhibition activity, and structural analysis suggested that the polysaccharides were arabinoxylan, featuring a xylan backbone with a majority of Araf and minor fractions of Manp, Galp, and Glcp involved in the side chain. The antifreeze arabinoxylan, characterized by lower molecular weight, less branching, and more flexible conformation, could weaken the hydrogen bonding of the surrounding water molecules more evidently, thus retarding the transformation of water molecules into the ordered ice structure.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Engineered ice-binding protein (FfIBP) shows increased stability and resistance to thermal and chemical denaturation compared to the wildtype.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing-thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.

Psychrophiles to control ice-water phase changes in frost-susceptible soils.

The phase changes of soil water or porous media have a crucial influence on the performance of natural and man-made infrastructures in cold regions. While various methods have been explored to address the impacts of frost-action arising from these phase changes, conventional approaches often rely on chemicals, mechanical techniques, and the reuse of waste materials, which often exhibit certain limitations and environmental concerns. In contrast, certain organisms produce ice-binding proteins (IBPs) or antifreeze proteins (AFPs) to adapt to low temperatures, which can inhibit ice crystal growth by lowering the freezing point and preventing ice crystallization without the need for external intervention. This study explores the potential of three psychrophilic microbes: Sporosarcina psychrophile, Sporosarcina globispora, and Polaromonas hydrogenivorans, to induce non-equilibrium freezing point depression and thermal hysteresis in order to control ice lens growth in frost-susceptible soils. We hypothesize that the AFPs produced by psychrophiles will alter the phase changes of porous media in frost-susceptible soils. The growth profiles of the microbes, the concentration of released proteins in the extracellular solution, and the thermal properties of the protein-mixed soils are monitored at an interval of three days. The controlled soil showed a freezing point of - 4.59 °C and thermal hysteresis of 4.62 °C, whereas protein-treated soil showed a maximum freezing point depression of - 8.54 °C and thermal hysteresis of 7.71 °C. Interestingly, except for the controlled sample, all the protein-treated soil samples were thawed at a negative temperature (minimum recorded at - 0.85 °C). Further analysis showed that the treated soils compared to porous media mixed soil freeze (1.25 °C vs. 0.51 °C) and thaw (2.75 °C vs. 1.72 °C) at extensive temperature gap. This freezing and thawing temperature gap is the temperature difference between the beginning of ice core formation and completed frozen, and the beginning of ice core thawing and completed thawed for the treated soil samples selected from different incubation days. Overall, this study presents a novel bio-mediated approach using psychrophilic microbes to control ice formation in frost-susceptible soils.

Characteristics and applications of plant-derived antifreeze proteins in frozen dough: A review.

Frozen dough technology has been widely used in the food industry at home and abroad due to its advantages of extending shelf life, preventing aging, and facilitating refrigeration and transportation. However, during the transportation and storage process of frozen dough, the growth and recrystallization of ice crystals caused by temperature fluctuations can lead to a deterioration in the quality of the dough, resulting in poor sensory characteristics of the final product and decreased consumption, which limits the large-scale application of frozen dough. In response to this issue, antifreeze proteins (AFPs) could be used as a beneficial additive to frozen dough that can combine with ice crystals, modify the ice crystal morphology, reduce the freezing point of water, and inhibit the recrystallization of ice crystals. Because of its special structure and function, it can well alleviate the quality deterioration problem caused by ice crystal recrystallization during frozen storage of dough, especially the plant-derived AFPs, which have a prominent effect on inhibiting ice crystal recrystallization. In this review, we introduce the characteristics and mechanisms of action of plant-derived AFPs. Furthermore, the application of plant-derived AFPs in frozen dough are also discussed.

Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein.

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

Synergistic Effect of Hyperactive Antifreeze Protein on Inhibition of Gas-Hydrate Growth by Hydrophobic and Hydrophilic Groups.

Antifreeze proteins (AFPs) are biodegradable inhibitors that effectively prevent the formation of natural gas hydrates that block pipelines. In this study, molecular dynamics simulations were employed to establish a kinetic model of the hyperactive insect antifreeze protein (Tenebrio molitor, TmAFP) and its mutants to inhibit the growth of sI natural methane hydrate. Simulations revealed that the hydrophobic and hydrophilic groups of threonine (Thr) residues at hydrate-binding sites played a synergistic role in binding hydrates. The hydrophobic groups anchored TmAFP to the hydrate surface through residues Thr39-Thr65 by migrating pendant hydrophobic methyl groups to the hydrate semicages. The hydrophilic groups stabilized TmAFP by hydrogen bonding with water molecules and integrating them into a quasi-hydrate structure, which more effectively inhibited hydrate growth. The results suggest that the hydrate growth inhibition is attributed to both the shape complementarity and the flexibility of binding residues. The synergy between hydrophobic and hydrophilic groups provides guidance for the design of more effective hydrate inhibitors.

Coexisting with Ice Crystals: Cryogenic Preservation of Muscle Food─Mechanisms, Challenges, and Cutting-Edge Strategies.

Cryopreservation, one of the most effective preservation methods, is essential for maintaining the safety and quality of food. However, there is no denying the fact that the quality of muscle food deteriorates as a result of the unavoidable production of ice. Advancements in cryoregulatory materials and techniques have effectively mitigated the adverse impacts of ice, thereby enhancing the standard of freezing preservation. The first part of this overview explains how ice forms, including the theoretical foundations of nucleation, growth, and recrystallization as well as the key influencing factors that affect each process. Subsequently, the impact of ice formation on the eating quality and nutritional value of muscle food is delineated. A systematic explanation of cutting-edge strategies based on nucleation intervention, growth control, and recrystallization inhibition is offered. These methods include antifreeze proteins, ice-nucleating proteins, antifreeze peptides, natural deep eutectic solvents, polysaccharides, amino acids, and their derivatives. Furthermore, advanced physical techniques such as electrostatic fields, magnetic fields, acoustic fields, liquid nitrogen, and supercooling preservation techniques are expounded upon, which effectively hinder the formation of ice crystals during cryopreservation. The paper outlines the difficulties and potential directions in ice inhibition for effective cryopreservation.

Accurate Prediction of Antifreeze Protein from Sequences through Natural Language Text Processing and Interpretable Machine Learning Approaches.

Antifreeze proteins (AFPs) bind to growing iceplanes owing to their structural complementarity nature, thereby inhibiting the ice-crystal growth by thermal hysteresis. Classification of AFPs from sequence is a difficult task due to their low sequence similarity, and therefore, the usual sequence similarity algorithms, like Blast and PSI-Blast, are not efficient. Here, a method combining n-gram feature vectors and machine learning models to accelerate the identification of potential AFPs from sequences is proposed. All these n-gram features are extracted from the K-mer counting method. The comparative analysis reveals that, among different machine learning models, Xgboost outperforms others in predicting AFPs from sequence when penta-mers are used as a feature vector. When tested on an independent dataset, our method performed better compared to other existing ones with sensitivity of 97.50%, recall of 98.30%, and f1 score of 99.10%. Further, we used the SHAP method, which provides important insight into the functional activity of AFPs.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

Self-Assembled Nanostructures of Homo-Oligopeptide as a Potent Ice Growth Inhibitor.

This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.

Accumulation of Antifreeze Proteins on Ice Is Determined by Adsorption.

Antifreeze proteins (AFPs) facilitate the survival of diverse organisms in frigid environments by adsorbing to ice crystals and suppressing their growth. The rate of AFP accumulation on ice is determined by an interplay between AFP diffusion from the bulk solution to the ice-water interface and the subsequent adsorption of AFPs to the interface. To interrogate the relative importance of these two processes, here, we combine nonequilibrium fluorescence experiments with a reaction-diffusion model. We find that as diverse AFPs accumulate on ice, their concentration in the aqueous solution does not develop a gradient but remains equal to its bulk concentration throughout our experiments. These findings lead us to conclude that AFP accumulation on ice crystals, which are smaller than 100 µm in radius, is not limited by the diffusion of AFPs, but by the kinetics of AFP adsorption. Our results imply that mass transport limitations do not hinder AFPs from performing their biological function.

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

MAIN CONCLUSION: Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17 °C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.