Ice Recrystallization Inhibition Research Articles

Tuning Ice Nucleation by Mussel-Adhesive Inspired Polyelectrolytes: The Role of Hydrogen Bonding

Tuning Ice Nucleation by Mussel-Adhesive Inspired Polyelectrolytes: The Role of Hydrogen Bonding

Preparation of Poly(vinyl Alcohol) Microparticles for Freeze Protection of Sensitive Fruit Crops.

Poly(vinyl alcohol) (PVA) displays ice recrystallization inhibition (IRI) properties as many antifreeze proteins found in cold tolerant organisms. The molecular architecture and composition (molecular weight and distribution of pendant OH and acetate groups) have been studied to improve the antifreezing properties of PVA, suggesting that the molecular architecture of PVA plays an important role in IRI activity. The present work deals with the preparation of PVA microparticles using an alkaline treatment. The effect of PVA molecular weight on the morphology and antifreezeing properties of PVA microparticles was investigated. The antifreezeing property of PVA microparticles on the susceptibility of flower bud tissues to freeze damage was also evaluated. The alkaline treatment of an aqueous PVA solution produced stable polymer chain aggregates with spherical shapes. The average size of the PVA microparticles increased significantly with the increasing molecular weight of the PVA macromolecule precursor. The PVA microparticles inhibited the growth of ice crystals and blocked ice growth at concentrations as low as 0.01 % w/v. The effect of impeding ice crystal growth by preventing the joining of adjacent ice crystals is attributed to the larger size of the PVA particles adsorbed on the ice surface compared to the aggregated PVA macromolecules in saline solution. The thermal hysteresis activity of PVA macromolecules and microparticles was not detected by differential scanning calorimetry analysis. The PVA microparticles reduced the incidence of freeze injuries in flower bud tissues by 55% and their application, considering the low toxicity of PVA, has a high potential for freeze protection in fruit crops.

Antifreezing Hydroxyl Monolayer of Small Molecules on a Nanogold Surface.

The rational design of ice recrystallization inhibition (IRI) materials is challenging due to the poor understanding of the IRI mechanism at the molecular level. Here we report several new findings about IRI. (1) A dense hydroxyl monolayer of small molecules, e.g. 6-aza-2-thiothymine (ATT), adsorbed on a nanogold surface was demonstrated, for the first time, to have IRI activity. Five structural analogues adsorbed on groups nanogold with outward hydroxyl or methyl were created to evidence the origin of IRI activity. (2) Their IRI mechanism is closely related to the density of hydroxyls on a nanogold surface. However, the hydrophobic interaction in our model is not essential for macroscopic IRI activity. (3) A molecular dynamics simulation elucidates the hydroxyl density dependent IRI trajectories underlying the experimental observations, and the radial distribution function reveals that the methyl even slightly hinders the formation of hydrogen bonding due to a hydrophobic interaction. This work sheds more light on the IRI mechanism that should help in the customization of novel IRI materials.

Comparative analysis of hydration layer reorientation dynamics of antifreeze protein and protein cytochrome P450

Antifreeze proteins (AFPs) inhibit ice re-crystallization by a mechanism remaining largely elusive. Dynamics of AFPs’ hydration water and its involvement in the antifreeze activity have not been identified conclusively. We herein, by simulation and theory, examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm, Choristoneura fumiferana, compared with a protein cytochrome P450 (CYP). The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules. Therefore, the jump reorientation of water molecules around the AFP active region is accelerated. Due to the mutual coupling and excitation of hydrogen bond exchange, with the acceleration of hydrogen bond exchange, the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated. Therefore, the water reorientation dynamics of AFP is faster than that of CYP. The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.

Bioinspired Cryoprotectants Enabled by Binary Natural Deep Eutectic Solvents for Sustainable and Green Cryopreservation

Cryopreservation plays an important role in prolonging the cell viability, but non-negligible cytotoxicity of cryoprotectants restrains its further development in many areas with safety concerns such as the food industry. Inspired by cold-tolerant animals, three binary natural deep eutectic solvents (NADESs) were prepared and used as cryoprotectants for achieving cytotoxicity-free and green cryopreservation, whose cryoprotective efficiency was compared with those of glycerol and dimethyl sulfoxide (DMSO), using yeast Saccharomyces cerevisiae, a common commercial microorganism in the food industry, as the cryopreservation object. Cytotoxicity evaluation involving reactive oxygen species accumulation and cell viability showed that the NADESs could be safe and biocompatible. In addition, cytomorphology indicated that the cells cryoprotected by NADESs show plump profiles with smooth cytoderms, while rough cytoderms or even severe cell distortion was partially observed for the ones cryopreserved by glycerol and DMSO. The live/apoptosis/death test of resuscitated cells based on flow cytometry illustrated that the NADES-cryopreserved cells could show higher cell viability (75–80%) with almost no apoptosis (<1.0%). Mechanisms of NADES-supported cryopreservation were further investigated concerning antifreezing capacities and ice recrystallization inhibition activities of NADESs, which suggested that NADESs could exhibit reinforced hydrogen-bonding strengths when decreasing temperature and correspondingly restrain the water molecular motion, making NADESs effectively inhibit ice recrystallization and prevent cells from ice-induced mechanical damage. The excellent cryoprotective efficiency of NADESs may help to open up new avenues for green cryopreservation and attract more concerns for NADESs as emerging cryoprotectants.

NMR Characterization of unfrozen brine vein distribution and structure in model packed beds

When a brine mixed with particles is frozen, some liquid water persists due to the freezing point depression caused by the solute impurity, surface energy, and disjoining pressure (wetting forces). This unfrozen water forms a complex “liquid vein network” (LVN). However, details of the freezing process are still not fully understood, including the permeability/tortuosity of the LVN, and the unfrozen water content at a given temperature. Here, we have applied nuclear magnetic resonance (NMR) relaxation, self-diffusion measurements and magnetic resonance imaging (MRI) to investigate the distribution and structure of LVNs. Magnesium chloride (MgCl 2 ) salt concentrations of 15, 30, and 60 mM were investigated with and without poly-methyl methacrylate (PMMA) particles of diameter 0.4, 9.9, and 102.2 μm, allowing us to quantify unfrozen water content and the structure of the LVN as a function of temperature, MgCl 2 concentration, and PMMA particle size. The results of magnetic resonance imaging (MRI) and self-diffusion confirm that the inhibition of ice recrystallization is a function of particle size. To gain information on LVN structure, we compared NMR results to Monte Carlo simulations of freezing in brine-particle systems. Comparisons between laboratory and simulation data suggest that, for our experimental range of temperature (−17.4 °C to −0.9 °C ± 0.5 °C), solutes make the dominant contribution to the unfrozen liquid fraction for particle sizes larger than a few microns, whereas in the finest grained porous media we tested, the unfrozen liquid fraction is controlled primarily by the films that wet particle-ice interfaces. • Non-invasive nuclear magnetic resonance techniques are applied to characterize the liquid vein network in frozen porous media. • The structure of liquid vein network and unfrozen water content are studied as a function of temperature, particle size and salt concentration. • The recrystallization process has been tracked by nuclear magnetic resonance techniques

Poly(l-alanine-co-l-lysine)-g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells.

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAKT) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAKT. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAKT and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAKT solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAKT solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAKT was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT. 1H NMR and FTIR spectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation.

Association mapping of autumn-seeded rye (Secale cereale L.) reveals genetic linkages between genes controlling winter hardiness and plant development

Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye (Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations (MTAs; p < 0.01) were revealed by association mapping. The ten most significant SNPs (p < 1.49e−04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, Chloroplast Unusual Positioning Protein-1 (CHUP1), FRIGIDA-like 4 (FRL4-like) protein, Chalcone Synthase 2 (CHS2), and Phenylalanine Ammonia-lyase 8 (PAL8). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions.

Ice Recrystallization Inhibition by Amino Acids: The Curious Case of Alpha- and Beta-Alanine

Extremophiles produce macromolecules which inhibit ice recrystallization, but there is increasing interest in discovering and developing small molecules that can modulate ice growth. Realizing their potential requires an understanding of how these molecules function at the atomistic level. Here, we report the discovery that the amino acid l-α-alanine demonstrates ice recrystallization inhibition (IRI) activity, functioning at 100 mM (∼10 mg/mL). We combined experimental assays with molecular simulations to investigate this IRI agent, drawing comparison to β-alanine, an isomer of l-α-alanine which displays no IRI activity. We found that the difference in the IRI activity of these molecules does not originate from their ice binding affinity, but from their capacity to (not) become overgrown, dictated by the degree of structural (in)compatibility within the growing ice lattice. These findings shed new light on the microscopic mechanisms of small molecule cryoprotectants, particularly in terms of their molecular structure and overgrowth by ice.

Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: effects of AF(G)Ps concentration and heat treatment.

This study compared ice recrystallization behaviors of frozen dessert model systems containing type I antifreeze protein (AFP I), type III antifreeze protein (AFP III), and antifreeze glycoprotein (AFGP) at -10 °C. Specifically, effects of AF(G)P concentration and heat treatment (95 °C for 10 min) were examined. The concentration dependence of the ice recrystallization rate constant reasonably well fit a sigmoidal function: the fitting procedure was proposed, along with cooperative coefficient α,and a new index of AF(G)P ice recrystallization inhibition (IRI) activity (C50). After 95 °C heat treatment for 10 min, AFP III lost its ice crystal recrystallization inhibitory activity the most: AFP I was less affected; AFGP was almost entirely unaffected. These different thermal treatment effects might reflect a lower degree of protein aggregation because of hydrophobic interaction after heat treatment or might reflect the simplicity and flexibility of the higher order structures of AFP I and AFGP.

Snow flea antifreeze peptide for cryopreservation of lactic acid bacteria

Cryogenic machining is one of the most commonly used techniques for processing and preserving in food industry, and traditional antifreeze agents cannot regulate the mechanical stress damage caused by ice crystals formed during recrystallization or thawing. In this study, we successfully developed an express system of a novel recombinant snow flea antifreeze peptide (rsfAFP), which has significant ice recrystallization inhibition ability, thermal hysteresis activity and alters ice nucleation, thus regulating extracellular ice crystal morphology and recrystallization. We showed that rsfAFP improved the survival rate, acid-producing ability, freezing stability, and cellular metabolism activity of Streptococcus thermophilus. We further showed that rsfAFP interacts with the membrane and ice crystals to cover the outer layer of cells, forming a dense protective layer that maintains the physiological functions of S. thermophilus under freezing stress. These findings provide the scientific basis for using rsfAFP as an effective antifreeze agent for lactic acid bacteria cryopreservation or other frozen food.

Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins.

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial. Here, we show that high IRI activity alone is insufficient to explain the beneficial effects of AF(G)Ps on human red blood cell (hRBC) survival. We show that AF(G)Ps with different IRI activities cause similar cell recoveries of hRBCs and that a modified AFGP variant with decreased IRI activity shows increased cell recovery. The AFGP variant was found to have enhanced interactions with a hRBC model membrane, indicating that the capability to stabilize cell membranes is another important factor for increasing the survival of cells after cryostorage. This information should be considered when designing novel synthetic cryoprotectants.

Inhibition of Ice Recrystallization by Nanotube-Forming Cyclic Peptides.

While most native ice-binding proteins are rigid, artificial (macro)molecular ice-binders are usually flexible. Realizing a regular array with precisely positioned ice-binding motifs on synthetic proteins, (macro)molecular ice-binders are thus challenging. Here, we exploit the predictable assembly of cyclic peptides into nanotubes as a starting point to prepare large, rigid ice-binders bearing an ice-binding site that is found in hyperactive ice-binding proteins in insects. First, we designed, synthesized, and purified cyclic octapeptide Lys2CP8 bearing a TaT motif to promote ice binding and investigated their solution assembly and activity using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, light scattering (LS), cryogenic transmission electron microscopy (cryo-TEM), and ice recrystallization inhibition (IRI) assays. The cyclic peptide Lys2CP8 was synthesized in good yield using Fmoc chemistry and purified by reversed-phase HPLC. Upon dissolution in aqueous solutions, Lys2CP8 was observed to assemble in a pH- and concentration-dependent manner into objects with nanoscopic dimensions. LS revealed the presence of small and large aggregates at pH 3 and 11, held together through a network of intermolecular antiparallel β-sheets as determined by FTIR and CD spectroscopy. Cryo-TEM revealed the presence of one-dimensional objects at pH 3 and 11. These are mostly well-dispersed at pH 3 but appear to bundle at pH 11. Interestingly, the pH-dependent self-assembly behavior translates into a marked pH dependence of IRI activity. Lys2CP8 is IRI-active at pH 3 while inactive at pH 11 hypothetically because the ice-binding sites are inaccessible at pH 11 due to bundling.

Inhibition of ice crystal growth by protein hydrolysates from different plant- and animal-based proteins

Due to the quality deterioration and the corresponding waste of frozen foods, there is a constant need for an effective, consumer and environmentally friendly, and sustainable anti-freezing ingredient. Hence, in this study, enzymatic hydrolysis and food grade chemical modification (succinylation) were evaluated to produce ice recrystallization inhibition (IRI) active peptide-based ingredients. Six different types of plant- and animal-based proteins were evaluated. Soy protein isolate (SPI), after 15- and 60- min alcalase hydrolysis, demonstrated a significant IRI activity compared to polyethylene glycol (PEG; as negative control) resulting in a two-third reduction of ice crystal size annealed at −8 ºC. Annealing temperature and peptide concentration also affected the IRI activity of SPI hydrolysate where decreased temperature and increased concentration resulted in a higher IRI activity. Increased hydrolysis time decreased the IRI activity of SPI hydrolysates but increased the IRI activity of sodium caseinate (SC) and pea protein isolate (PPI) hydrolysates. SPI-60 was less temperature resistant compared to SPI-15 which inhibited the growth of ice crystals (smaller than 23 µm). Moreover, chemical modification via succinylation reaction with OSA enhanced the IRI activity of the PPI and SPI hydrolysates leading to a 55.3% and 60.6% reduction in the size of ice crystals, respectively. Overall, this study demonstrates a great potential in the utilization of common food proteins for the production of value-added natural anti-freezing ingredients which could benefit the food industry by enhancing frozen foods’ shelf-life and reducing waste.

Macrocycle molecule-based cryoprotectants for ice recrystallization inhibition and cell cryopreservation

Cyclodextrin-based cryoprotectants were developed. α-TMCD, which can be easily put into large-scale production, showed enhanced cell viabilities of 19.97 ± 0.78%, 13.93 ± 4.46% and 19.10 ± 0.95% against GES-1, hucMSCs and A549 cells. Moreover, the viable cells observed by light microscope imaging showed that the enhanced hucMSC cell number percentage of α-TMCD was 103.2%. An α-TMCD-DMSO-based CPA exhibited an enhanced cryoprotective effect by a mechanism of DMSO-enhanced cell penetrating effect and α-TMCD-DMSO synergistically enhanced IMA ability. α-TMCD exhibited potential for the discovery of macrocycle-molecule-based cryoprotectants.

Potent Time-Dependent Ice Recrystallization Inhibition Activity of Cellulose Nanocrystals in Sucrose Solutions.

Exploring novel materials with ice recrystallization inhibition (IRI) activity in several fields often starts with a quantitative analysis of ice crystal size change by a splat assay or sandwich assay on a short time scale from 0.5 to 1 h. This study found that this time scale was insufficient to evaluate the IRI activity of cellulose nanocrystals (CNCs) in a model ice cream system-25.0% sucrose solution. No IRI activity was observed in CNCs incubated with ice crystals on a short time scale of 0.5-2.0 h. However, over longer time scales, the growth of ice crystals was entirely inhibited by 1.0% CNCs (between 2 and 24 h) and 0.5% CNCs (between 24 and 72 h) with corresponding final crystal sizes of 25 and 40 μm, respectively. Additionally, ice shaping was observed on a long exposure time, but not on a short exposure time. The findings presented here can be explained by a time-dependent surface coverage of CNCs on ice crystals. The data here indicate the importance of choosing a suitable exposure time for evaluating the IRI activity of new materials and prompt a better understanding of IRI mechanisms involving CNCs.

Isolation of novel wheat bran antifreeze polysaccharides and the cryoprotective effect on frozen dough quality

Different wheat bran antifreeze polysaccharides (WBAP) were first isolated by ice shell separation method. The composition and antifreeze activity of varied WBAP were characterized and their cryoprotective effects on frozen dough were comprehensively studied with the emphasis on ice crystals, yeast and gluten proteins. WBAP with enriched arabinoxylan (AX) of high arabinofuranose/xylopyranose ratio (A/X) showed superior effect on inhibiting ice recrystallization compared with WBAP with low A/X. WBAP could significantly improve the quality of frozen dough steamed bread, and the effect coincided with antifreeze activity. WBAP effectively inhibited the ice recrystallization and altered the shape of ice crystal from cubic to lamellate in frozen dough, consequently better preserving the yeast viability and dough structure. WBAP alleviated the depolymerization of glutenin macropolymers, and was conductive in developing more homogeneous and less distorted gluten network structures in frozen dough. The current study can provide a theoretical basis for the exploitation of WBAP as a technofunctional improver for frozen dough. • Wheat bran antifreeze polysaccharides (WBAP) with varied structures was isolated. • WBAP possessed thermal hysteresis activity and inhibited ice recrystallization. • The quality of frozen dough steamed bread was significantly improved by WBAP. • WBAP preserved yeast viability and dough structure by affecting ice growth and shape. • WBAP alleviated depolymerization of glutenin macropolymers in frozen dough.

The effects of selected stabilizers addition on physical properties and changes in crystal structure of whey ice cream

Dry sweet whey was used in this study at 42% to prepare a new ice cream composition. The additional aim of the research was to select the most effective stabilizers in ice recrystallization inhibition. The addition and impact of two stabilizing systems, containing LBG (locust bean gum), guar gum and respectively κ- and ι-carrageenan, on physicochemical properties (density, pH, overrun, viscosity, melting time) and crystal structure of ice cream after a specified storage time of: 24 h, 1 month and 3 months were examined. It was found that whey presence increases the density and viscosity and reduces the overrun of the analyzed product. Addition of stabilizing mixes also differentiated density (higher for the samples with additives), melting time and overrun. The lowest overrun value and simultaneously the shortest melting time were recorded for ι-carrageenan addition. No influence of stabilizing systems on pH of examined ice cream was found. The stabilizing mix containing ι-carrageenan was found to be the best in recrystallization inhibition, demonstrated by the presence of crystals with the smallest diameter 28 μm, after 3 months of storage, while for the sample without additives 68 μm was noted.

Beetle and mussel-inspired chimeric protein for fabricating anti-icing coating

Ice accretion on surfaces can cause serious damages and economic losses in industries and civilian facilities. Antifreeze proteins (AFPs) as evolutionary adaptation products of organisms to cold climates, provide solutions for alleviating icing problems. In this work, a chimeric protein Mfp-AFP was rationally designed combining mussel-inspired adhesive domain with Tenebrio molitor-derived antifreeze protein domain. Expectedly, the multifunctional Mfp-AFP can lower the freezing point of water and inhibit ice recrystallization. The chimeric protein could also readily modify diverse solid surfaces due to the adhesive domain containing Dopa, and resist frosting and delay ice formation due to the beetle-derived antifreeze fragment. Moreover, Mfp-AFP coatings display excellent biocompatibility proved by cytocompatibility and hemolysis assays. Here, the designed multifunctional protein coatings provide an alternative strategy for fabricating anti-icing surfaces.

Design of an Ice Recrystallization-Inhibiting Polyampholyte-Containing Graft Polymer for Inhibition of Protein Aggregation.

Freezing-induced damage to proteins, through osmotic stress and ice recrystallization, during protein processing and long-term storage is a serious concern and may lead to loss of protein activity owing to denaturation. In this study, graft copolymers composed of a cryoprotective polymer (capable of preventing osmotic stress) and poly(vinyl alcohol) (PVA; known for its high ice recrystallization inhibition (IRI) property) were developed. The polymers had high IRI activity, albeit slightly lower than that of PVA alone, but substantially higher than that of succinylated ε-poly-l-lysine (PLLSA) alone. The graft polymers showed an efficiency higher than that of PVA or PLLSA alone in protecting proteins from multiple freeze-thaw cycles, as well as during prolonged freezing, indicating a synergy between PVA and PLLSA. The PLLSA-based graft polymer is a promising material for use in protein biopharmaceutics for the long-term storage of proteins under freezing conditions.