Antifreeze Proteins Research Articles

Effect of staged thawing assisted antifreeze protein on quality of repeatedly freeze-thawed minced pork

The effects of thawing at 4 °C, 25 °C, 25/-1 °C (staged thawing) on the quality of minced pork added with 0.2% antifreeze protein (AFP) and without AFP were respectively assessed by investigating the total thawing time, material loss, water distribution and Magnetic Resonance Imaging (MRI), color, microstructure, bacterial count, and lipid and protein oxidation of minced pork in the study. The results showed that the total thawing time of minced pork thawed at 25/-1 °C was significantly lower than that of thawed at 4 °C (P < 0.05). The material loss of the AFP sample thawed at 25/-1 °C was reduced. Meanwhile, the AFP samples thawed at 25/-1 °C appeared the shortest T22 relaxation time at the same F-T cycles, and inhibited the oxidation of lipid and protein, presenting the lowest malondialdehyde content and the highest the total sulfhydryl content, and restrained destruction of minced pork microstructure and slowed down the growth of microorganism during freeze-thaw cycles. Summarily, thawed at 25/-1 °C combined with 0.2% AFP addition emerged a tremendous potential to improve the quality of the repeatedly frozen-thawed pork.

Improving Antifreeze Proteins Prediction with Protein Language Models and Hybrid Feature Extraction Networks.

Accurate identification of antifreeze proteins (AFPs) is crucial in developing biomimetic synthetic anti-icing materials and low-temperature organ preservation materials. Although numerous machine learning-based methods have been proposed for AFPs prediction, the complex and diverse nature of AFPs limits the prediction performance of existing methods. In this study, we propose AFP-Deep, a new deep learning method to predict antifreeze proteins by integrating embedding from protein sequences with pre-trained protein language models and evolutionary contexts with hybrid feature extraction networks. The experimental results demonstrated that the main advantage of AFP-Deep is its utilization of pre-trained protein language models, which can extract discriminative global contextual features from protein sequences. Additionally, the hybrid deep neural networks designed for protein language models and evolutionary context feature extraction enhance the correlation between embeddings and antifreeze pattern. The performance evaluation results show that AFP-Deep achieves superior performance compared to state-of-the-art models on benchmark datasets, achieving an AUPRC of 0.724 and 0.924, respectively.

Effect of antifreeze protein I in the freezing solution on in vivo-derived sheep embryos

This study evaluated the effects of different antifreeze protein type I (AFP I) concentrations added to a slow freezing solution in sheep in vivo-derived embryos. Good-quality embryos were allocated into: AFP-free (CONT); 0.1 μg/mL of AFP I (AFP0.1); or 0.5 μg/mL of AFP I (AFP0.5). After thawing, embryos were in vitro cultured (IVC) for 48 h. At 24 h and 48 h of IVC, dead cells and apoptosis, mitochondrial activity, intracellular reactive oxygen species (ROS), and glutathione (GSH) evaluations were performed. At 24 h, evaluated embryos were submitted to RT-qPCR for metabolism (SIRT2, PRDX1, OCT4, CDX2) and quality (AQP3, CDH1, HSP70, BAX, BCL2) genes. The in vitro survival rate was 56% (22/39) for CONT, 60% (32/53) for AFP0.1, and 53% (23/43) for AFP0.5 (p > 0.05). A tendency (p = 0.09) for a higher blastocyst hatching rate was noted in AFP0.1 (62%) compared to AFP0.5 (33%), and both groups were similar to CONT (50%). An increased (p < 0.05) mitochondrial activity at 24 h was observed in AFP0.1 compared to CONT. No differences (p > 0.05) were observed in oxidative stress homeostasis and viability between treatments. A downregulation (p < 0.05) of CDH1 in AFP0.1 and a downregulation of AQP3 in AFP0.5 were observed in comparison to the other groups. An upregulation (p < 0.05) was detected in HSP70 and BCL2 on AFP0.5 compared to AFP0.1 group. The addition of AFP I in slow freezing solution can benefit cryopreserved sheep in vivo-derived embryos, without affecting embryonic survival.

MXene, protein, and KCl-assisted ionic conductive hydrogels with excellent anti-freezing capabilities, self-adhesive, ultra-stretchability, and remarkable mechanical properties for a high-performance wearable flexible sensor.

Developing a hydrogel with switchable features and freeze tolerance is remarkably significant for designing flexible electronics to adjust various application needs. Herein, MXenes, AFPs (antifreeze proteins), and potassium chloride (KCl) were introduced to a polyacrylamide (PAM) polymer network to design an anti-freezing hydrogel. The ionic hydrogels are characterized by excellent ionic conductivity, presenting adjustable properties of remarkable mechanical strength and self-adhesion to meet individualized application demands. The capability of KCl and AFPs to inhibit ice crystals gives hydrogels with anti-icing properties under a low-temperature atmosphere. The PAM/MXene15/AFP30/KCl15 hydrogel demonstrated negligible hysteresis behavior, quick electromechanical response (0.10 s), and excellent sensitivity (gauge factor (GF) = 13.1 within the strain range of 1200-2000%). The resulting hydrogel could be immobilized on the animal or human skin to detect different body movements and physiological motions, offering reproducibility and precise accuracy as primary advantages. The approach of developing materials with tunable features, along with inorganic salt and the fish-inspired freeze-tolerance method, offers a new prospect for wearable gadgets.

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

Research Progress on Plant Anti-Freeze Proteins

Research Progress on Plant Anti-Freeze Proteins

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Recent advances, challenges and functional applications of antifreeze protein in food industry

SummaryFreezing is a common food preservation method that can extend the shelf life of food, but the ice crystals and recrystallisation during freezing, storage and transportation cause significant damage to frozen food. Antifreeze proteins are a class of proteins that widely exist in organisms living in cold conditions, such as fish, insects, plants and microorganisms in Antarctica and other cold regions. Antifreeze proteins can reduce the freezing point of water, inhibit recrystallisation effects, modify ice crystal morphology and effectively suppress the quality deterioration caused by ice crystals during food freezing. This review comprehensively introduces the source, antifreeze mechanism, application in food, influencing factors and regulating methods of antifreeze activity of antifreeze proteins, as well as the limitations of antifreeze proteins. It also looks forward to the future application of antifreeze proteins in food.

Molecular insights into the wettability of antifreeze protein-adsorbed hydrate surface: Application to hydrate anti-agglomeration

Adding kinetic inhibitors (KIs) to prevent hydrate formation or anti-agglomerate agents (AAs) adsorbed on hydrate crystals to suppress particle aggregation are both effective means to eliminate hydrate blockage in pressurized pipelines. Antifreeze proteins (AFPs) are representative KIs that hinder hydrate growth via the interfacial adsorption mechanism. This implies the possibility of interfacial modification and simultaneous anti-aggregation effects, which yet have not been explored. Here molecular dynamics (MD) simulations were employed to determine the possible anti-aggregation performance of AFP. Specifically, we identified larger contact angles, that is, the enhanced hydrophobicity, of AFP-adsorbed CH4 hydrate under the dodecane oil phase. This indicates the possible dual inhibition effects of AFP to ensure oil transportation. However, no significant effects of AFP on wettability were observed in the systems with the CO2 hydrate substrate or gas environment. This contrasting behavior was explained by the differences in the hydrogen-bonding formation and interaction strength between substrates and water droplets, which are strongly influenced by the charged residues of AFP. Additionally, we compared AFP with traditional surfactants and emphasized that the anti-aggregation abilities of additives are intricately linked to their functional group arrangements and inherent structures. Our work provides molecular-level guidance for designing effective hydrate inhibitors with dual performance to ensure flow assurance.

39 The effect of the supplementation of type III antifreeze protein on cryotolerance of bovine in vitro-produced blastocysts

39 The effect of the supplementation of type III antifreeze protein on cryotolerance of bovine in vitro-produced blastocysts

Metabolomic insights into the effect of chickpea protein hydrolysate on the freeze–thaw tolerance of industrial yeasts

The use of frozen dough is an intensive food-processing practice that contributes to the development of chain operations in the bakery industry. However, the fermentation activity of yeasts in frozen dough can be severely damaged by freeze–thaw stress, thereby degrading the final bread quality. In this study, chickpea protein hydrolysate significantly improved the quality of steamed bread made from frozen dough while enhancing the yeast survival rate and maintaining yeast cell structural integrity under freeze–thaw stress. The mechanism underlying this protective role of chickpea protein hydrolysate was further investigated by untargeted metabolomics analysis, which suggested that chickpea protein hydrolysate altered the intracellular metabolites associated with central carbon metabolism, amino acid synthesis, and lipid metabolism to improve yeast cell freeze–thaw tolerance. Therefore, chickpea protein hydrolysate is a promising natural antifreeze component for yeast cryopreservation in the frozen dough industry.

Bioinspired durable interpenetrating network anti-icing coatings enabled by binders and hydrophobic-ion specific synergies

Designing a coating with a comprehensive range of anti-icing properties is a difficult challenge in the current complex and variable icing environment. Bionic anti-icing coatings have received widespread attention in recent years, but their exposure to mechanical damage conditions tends to result in insufficient durability. This work proposes a novel approach inspired by the antifreeze proteins (AFPs) found in fish, wherein a durable interpenetrating coating with excellent anti-icing and deicing performance is designed by combining a fluorinated amphiphilic ionic polymer with a biomimetic binder. The incorporation of hydrophobic fluorinated chain segments and amphiphilic ionic chain segments effectively inhibits ice nucleation (DT = 2010 s, TIN = − 22.1 °C), slows down ice propagation, and reduces ice adhesion (τ = 22.2 kPa) through the synergistic effect of hydrophobicity and ionic specificity. Furthermore, the addition of a bionic binder to create an interpenetrating network enhances the mechanical properties and anti-icing durability of the coating. Moreover, it can further function as anti-fogging, self-cleaning, and antibacterial for glass surfaces, demonstrating significant potential in the new generation of optical and medical devices.

Another gift from nature to human beings: himalayan antifreeze protein that acts as a cryoprotectant

Another gift from nature to human beings: himalayan antifreeze protein that acts as a cryoprotectant

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.

Natural self-assembled supramolecules as green inhibitors against methane hydrate formation

Environmentally friendly hydrate inhibitors are the most promising method to solve the environmental pollution of inhibitors, among which Safranine O (SO) with antifreeze protein properties shows excellent application prospects. SO will self-assemble into macromolecules under hydrogen bonding and electrostatic action in an aqueous solution. The assembled macromolecules' hydrophobic and hydrophilic functional groups are regularly arranged with the properties of an antifreeze protein. In the crystal growth inhibition (CGI) test, the supercooling degree of the complete inhibition region (CIR) of 1.0 wt% SO solution for SI hydrate was significantly higher than that of 1.0 wt% PVP solution. Further, the acidic environment did not affect the inhibitory effect of SO on hydrate. The complete inhibition range of 1.0 wt% SO solution on SI hydrate at pH 1.4 and 7.1 was 7.41 °C and 8.35 °C, respectively. These results suggest that SO-based KHIs may provide new ideas for developing highly efficient acid-resistant hydrate inhibitors.

Uniformly Hybrid Surface Containing Adjustable Hydrophobic/Hydrophilic Components Obtained by Programmed Strain for Synergistic Anti-Icing.

Sufficient efforts have been put into the design of anti-icing materials to eliminate the icing hazard. Among the currently approved anti-icing concepts, hydrophilic/hydrophobic hybrid anti-icing materials inspired by antifreeze proteins show excellent properties in inhibiting ice nucleation, inhibiting ice crystal growth, and reducing ice adhesion. However, it is still a great challenge to accurately regulate the hydrophilic and hydrophobic hybrid components of the coating surface to clarify the synergistic mechanism. This work proposes a strain-manipulated surface modification strategy, and an anti-icing coating with adjustable hydrophilic/hydrophobic hybrid components prepared by combining chemical vapor deposition and siloxane chemistry is obtained. According to the ice resistance experiment at -15 °C, the performance of anti-icing is closely related to the proportion of hydrophilic and hydrophobic hybrids. The icing delay time and ice adhesion strength of the material with the optimal hydrophilic/hydrophobic components are 280 s and 18.6 kPa, respectively. These unique properties can be attributed to the synergistic effect of hydrophilic and hydrophobic structures on the regulation of interfacial water.

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.

Bio‐Inspired Sandwich‐Structured All‐Day‐Round Solar Evaporator for Synergistic Clean Water and Electricity Generation

AbstractThe integration of solar‐driven interfacial evaporation and electricity co‐generation is considered a promising approach to simultaneously alleviate freshwater scarcity and the energy crisis. However, affected by intermittent solar irradiation/uncontrollable weather, the overall performance of solar‐driven evaporation in the real world is greatly reduced. Herein, inspired by antifreeze proteins in beetles that survive in extreme climates, all‐weather solar‐driven interfacial evaporators with a sandwich structure are designed. The top and bottom layers composed of MnO2‐modified cotton cloth are used for photothermal conversion and water transport, meanwhile, the middle layer made of a phase change microcapsule/hydrogel composite serves for heat storage and release. Under 1 kW m−2 irradiation, the evaporator exhibits a high evaporation rate of 2.67 kg m−2 h−1 and an efficiency of 89.5%. In the dark, the heat released from the phase change layer supports an evaporation rate of 0.43 kg m−2 h−1, 3.6 times that of pure water. Additionally, assembled with a thermoelectric module, the hybrid device achieves a stable output electricity power of 0.42 W m−2 under 1‐sun illumination and a prolonged output for 30 min in the dark. This work provides a novel approach for full‐time solar‐powered steam‐electricity co‐generation and a proof of concept for biomimetic steam generation/heat management integration.

Whole-genome sequence and mass spectrometry study of the snow blight fungus Phacidium infestans (Karsten) DSM 5139 growing at freezing temperatures

Phacidium infestans (synonym Gremmenia infestans) is a significant pathogen that impacts Pinus species across the northern regions of Europe and Asia. This study introduces the genome sequence of P. infestans Karsten DSM 5139 (Phain), obtained through Pacbio technology. The assembly resulted in 44 contigs, with a total genome size of 36,805,277 bp and a Guanine–Cytosine content of 46.4%. Genome-mining revealed numerous putative biosynthetic gene clusters that code for virulence factors and fungal toxins. The presence of the enzyme pisatin demethylase was indicative of the potential of Phain to detoxify its environment from the terpenoid phytoalexins produced by its host as a defense mechanism. Proteomic analysis revealed the potential survival strategies of Phain under the snow, which included the production of antifreeze proteins, trehalose synthesis enzymes, desaturases, proteins related to elongation of very long-chain fatty acids, and stress protein responses. Study of protein GH11 endoxylanase expressed in Escherichia coli showed an acidic optimum pH (pH 5.0) and a low optimum temperature (45 °C), which is reflective of the living conditions of the fungus. Mass spectrometry analysis of the methanol extract of Phain, incubated at − 3 °C and 22 °C, revealed differences in the produced metabolites. Both genomic and mass spectrometry analyses showed the ability of Phain to adapt its metabolic processes and secretome to freezing temperatures through the production of osmoprotectant and cryoprotectant metabolites. This comprehensive exploration of Phain's genome sequence, proteome, and secretome not only advances our understanding of its unique adaptive mechanisms but also expands the possibilities of biotechnological applications.

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.