Ice Recrystallization Inhibition Activity Research Articles

Adsorption of ice-binding proteins onto whole ice crystal surfaces does not necessarily confer a high thermal hysteresis activity

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called “thermal hysteresis (TH)”, and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called “ice recrystallization inhibition (IRI)”. To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.

Effects of polysaccharides autoclave extracted from Flammulina velutipes mycelium on freeze-thaw stability of surimi gels

Quality deterioration caused by ice formation and growth is a prevailing problem in frozen food, therefore, exploration of natural cryoprotectants has been attracting more attention. In this study, Flammulina velutipes mycelium polysaccharides (FVP) were obtained by autoclave extraction and their effect on freeze-thaw stability of surimi gels were investigated. The results showed that FVP consisted mainly (59.02%) of high-molecular-weight polysaccharides (>450 kDa), and were composed of Glu, Xyl, Man, Gal, and Fuc in a molar ratio of 33.4: 28.72: 26.31: 8.01: 3.55. FVP (1.0 mg/mL) showed superior Ice Recrystallization Inhibition (IRI) activity. Freeze-thaw stability tests demonstrated that frozen-thawed surimi gels with FVP addition exhibited a more uniform and continuous matrices than the control ones. FVP could significantly improve the water holding capacity, the hardness, springiness and cohesiveness of surimi gels during the repeated freeze-thaw cycles. Studies on water state and properties showed that FVP could decrease the freezable water content, restrict water mobility, and inhibit water migration in frozen surimi gels. These results suggest the potential of FVP as a promising cryoprotectant candidate in the frozen food industry.

Biomimetic ice recrystallization inhibition-active poly(vinyl alcohol) enhances the freeze-thaw resistance of cement paste

This work presents experimental evidence that specific molecular weights of poly(vinyl alcohol) (PVA), a molecule that displays biomimetic ice recrystallization inhibition (IRI) behavior, are required to impart freeze-thaw resistance to portland cement paste when PVA is added as a water-solubilized admixture. Three molecular weights of PVA were first tested for IRI activity. IRI-active PVA-modified cement pastes were then prepared and tested for freeze-thaw resistance. X-ray micro-computed tomography was employed to investigate internal damage and to characterize the air void system. Differential scanning calorimetry was used to quantify and compare the amount of ice that formed in the hardened pastes. Mercury intrusion porosimetry was used to characterize the pore-size distributions of cement pastes. Results substantiate that PVA with a molecular weights >31,000 g/mol can impart freeze-thaw resistance to cement paste and that the freeze-thaw enhancement is not due to improved hydrophobicity or an adequately entrained air void system. PVA additions did lead to reductions in porosity. However, the reductions were insufficient to fully explain why lower quantities of ice formed during freezing, indicating that IRI-active biomimetic polymers, like PVA, can enhance the freeze-thaw resistance of cement paste due, in part, to their interaction with ice.

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.

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.

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.

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.

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.

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.

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.

Surfactant properties of a biomimetic antifreeze polymer admixture for improved freeze-thaw durability of concrete

Recently, a novel bioinspired approach for improving freeze–thaw resistance of concrete was proposed as an alternative to traditional air entrainment. The mechanism is based on ice recrystallization inhibition (IRI) activity displayed by antifreeze proteins and synthetic, biomimetic replicates, like nonionic polyethylene glycol–graft–polyvinyl alcohol (PEG-PVA), which can be added as a polymeric admixture to concrete in the fresh state. To further substantiate the mechanistic underpinnings of the air-entraining agent (AEA) alternative, this work measures and reports the foam stability and interfacial surface tension properties of nonionic PEG-PVA. Foam stability and tensiometry measurements of PEG-PVA were quantified and compared to a conventional anionic AEA, MasterAir (MA). Results demonstrate that PEG-PVA requires much higher concentrations (0.77–2.8% by weight of cement) to form a stable foam system in comparison to the AEA (0.027–0.19%). The results correlated well with foam stability measurements, which showed PEG-PVA’s inability to form long-lasting stable foams in comparison to MA. Tensiometry measurements also revealed that high concentrations (10–60 g/L) of PEG-PVA were required to reduce the surface tension of water in comparison to MA (0.015–0.045 g/L), indicating that PEG-PVA neither acts as a surfactant nor readily self-assembles into micelles at concentrations that proved to provide freeze–thaw resistance. Nevertheless, PEG-PVA-modified concrete showed similar resistance to MA-modified concrete in freeze–thaw cycling at concentrations as low as 0.066% by weight of cement while entraining only minimal amounts of air, suggesting that the freeze–thaw resistance is due at least in part to the IRI activity of PEG-PVA instead of its ability to entrain a proper air void system.

Poly(l-Ala-co-l-Lys) Exhibits Excellent Ice Recrystallization Inhibition Activity.

The control of ice recrystallization is very important in cryo-technological fields such as the food industry, biopharmaceuticals, and cell storage. Ice recrystallization inhibition (IRI) compounds are therefore designed to limit the growth of ice crystals, decrease the crystal size, and control the crystal shape. To improve the IRI activity of cryo-systems, various synthetic polymers such as biomimetic polypeptides from polar fish, facially amphiphilic polymers, polyampholytes, poly(vinyl alcohol) derivatives, and block copolymers with hydrophilic-hydrophobic balance have been developed. Except for graphene oxide, poly(vinyl alcohol) has thus far exhibited the best performance among these polymers. Herein, poly(l-alanine-co-l-lysine) (PAK) was shown to exhibit a similar IRI activity to that of poly(vinyl alcohol). Moreover, in contrast to the needle-shaped ice crystals generated by the aqueous PVA solution, the PAK solution was shown to generate cubic-to-spherical shaped ice crystals. Furthermore, neither poly(l-alanine-co-l-aspartic acid) (PAD) nor poly(ethylene glycol) (PEG) with a similar molecular weight provided any significant IRI activity. Examination by FTIR and circular dichroism spectroscopies indicated that the PAK forms α-helices, whereas the PAD forms random coils in water. Further, a dynamic ice shaping study suggested that PAK strongly interacts with ice crystals, whereas PAD and PEG only weakly interact. These results suggest that PAK is an important compound with superior IRI activity and that this activity is dependent upon the functional groups and secondary structure of the polypeptides.

Size and Shape Control of Ice Crystals by Amphiphilic Block Copolymers and Their Implication in the Cryoprotection of Mesenchymal Stem Cells.

Precise control over the size and shape of ice crystals is a key factor to consider in designing antifreezing and cryoprotecting molecules for cryopreservation of cells. Here, we report that a poly(ethylene glycol)-poly(l-alanine) (PEG-PA) block copolymer exhibits excellent cryoprotecting properties for stem cells and antifreezing properties for water. As the molecular weight of PA increased from 500, 760, and 1750 Da (P1, P2, and P3) at the same PEG molecular weight of 5000 Da, the β-sheet content decreased and α-helix content increased. Comparing P2 (PEG-PA; 5000-760) and P4 (PEG-PA: 1000-750), β-sheets increased as the PEG block length decreased. The critical micelle concentration of the PEG-PA block copolymers was in a range of 0.5-3.0 mg/mL and was proportional to the hydrophobicity of the PEG-PA block copolymers. The P1, P2, and P3 self-assembled into spherical micelles, whereas P4 formed micelles with cylindrical morphology. The difference in the block copolymer structure affected ice recrystallization inhibition (IRI) activity and cryopreservation of cells. IRI activity was assayed via mean largest grain size (MLGS), and interactions between polymers and ice crystal surfaces were studied by dynamic ice-shaping studies. The MLGS decreased to 58 → 53 → 45 → 35 → 23% of that of PBS, as the polymer (PEG-PA 5000-500) concentration increased from 0.0 (PBS; control) → 1.0 → 5.0 → 10 → 30 → 50 mg/mL. The MLGS of PEG 5k solutions (negative control) decreased to 74 → 71 → 64 → 44 → 37% of that of PBS in the same concentration range. P3 and P4 with a longer hydrophobic PA block developed elongated ice crystals at above 30 mg/mL. The dynamic ice-shaping study exhibited that ice crystals became needle-shaped, as the hydrophobicity of the polymer increased as in P2-P4. The cell recovery in the P1 system after cryopreservation at -196 °C for 7 days was 87% of that of the dimethyl sulfoxide (DMSO) 10% system (positive control). The cell recovery was 48% for the P2 system and drastically decreased to less than 30% of that of the DMSO 10% system in the P3, P4, PEG 5k, PEG 1k, PVA 80H, and PVA 100H systems. Current studies suggest that IRI activity, round ice crystal shaping, and membrane stabilization activity of P1 cooperatively provide excellent cell recovery among the candidate systems. Recovered stem cells exhibited excellent proliferation and multilineage differentiation into osteocytes, chondrocytes, and adipocytes. To conclude, the PEG-PA (5000-500) block copolymer is suggested to be a promising antifreezing cryoprotectant for stem cells.

Ice recrystallization inhibition effect of cellulose nanocrystals: Influence of sucrose concentration

Stabilizers are commonly used to inhibit the ice recrystallization process that leads to the formation of large ice crystals, decreasing consumer acceptance of ice cream. However, the ice recrystallization inhibition (IRI) effect of stabilizers is very dependent on measurement conditions. The cause of this dependence is not clear, which imposes problems in comparing the IRI activity and understanding the IRI mechanism of stabilizers. This study investigated the influence of sucrose concentration on the IRI effect of cellulose nanocrystals (CNCs), which are newly identified IRI active materials. A splat assay and a sandwich assay were used to determine IRI activities of CNCs at sucrose concentrations from 2.0% to 49.0% w/w and incubation temperature of −8 °C for 30 min. The IRI activities of CNCs were higher at low sucrose concentrations, almost disappeared at medium sucrose concentrations, and restored slightly at high sucrose concentrations. The IRI effect of CNCs is correlated with the ratio between CNCs concentration in the unfrozen phase and total surface area of ice crystals. This surface area-related IRI effect was also observed with guar gum, locust bean gum, and poly(vinyl alcohol). It is a strong evidence of an ice-binding mechanism for the IRI effect of stabilizers. These research findings offer a new perspective in comparing and understanding the measurement condition-dependent IRI effect of stabilizers, which could potentially benefit the ice cream industry in developing new materials and recipes to control ice recrystallization.

Methods in Biosynthesis and Characterization of the Antifreeze Protein (AFP) for Potential Blood Cryopreservation

The cryopreservation of red blood cells (RBCs) is very important to modern medicine. Cryoprotectants (CPAs) such as dimethyl sulfoxide (DMSO), proline, trehalose, and polyvinyl alcohol (PVA) have been used in the cryopreservation of RBCs, but the results are not satisfactory. Marinomonas primoryensis antifreeze protein (MpAFP) is a Ca2+-dependent AFP derived from Antarctic bacteria, which can prevent bacteria from freezing under extremely cold conditions and may be suitable for cryopreservation of RBCs. The active region of MpAFP is located in region IV and is called MPAFP_RIV. In this paper, the gene of region IV of MpAFP is introduced into BL21 (DE3) competent cells, and MpAFP_RIV is obtained after culture, separation, and purification. The improved splat assay is proposed, and this method first proves that MpAFP_RIV has strong ice recrystallization inhibition (IRI) activity without Ca2+. The improved splat assay is easier to operate and lower the cost compared to the traditional one, and the results are consistent with the classic sucrose sandwich assay, proving that this method can accurately detect IRI activity. The feasibility of MPAFP_RIV combined with classical CPA for cryopreservation of RBCs and the methods to increase the yield of MPAFP_RIV are proposed.

Polymer Self-Assembly Induced Enhancement of Ice Recrystallization Inhibition.

Ice binding proteins modulate ice nucleation/growth and have huge (bio)technological potential. There are few synthetic materials that reproduce their function, and rational design is challenging due to the outstanding questions about the mechanisms of ice binding, including whether ice binding is essential to reproduce all their macroscopic properties. Here we report that nanoparticles obtained by polymerization-induced self-assembly (PISA) inhibit ice recrystallization (IRI) despite their constituent polymers having no apparent activity. Poly(ethylene glycol), poly(dimethylacrylamide), and poly(vinylpyrrolidone) coronas were all IRI-active when assembled into nanoparticles. Different core-forming blocks were also screened, revealing the core chemistry had no effect. These observations show ice binding domains are not essential for macroscopic IRI activity and suggest that the size, and crowding, of polymers may increase the IRI activity of “non-active” polymers. It was also discovered that poly(vinylpyrrolidone) particles had ice crystal shaping activity, indicating this polymer can engage ice crystal surfaces, even though on its own it does not show any appreciable ice recrystallization inhibition. Larger (vesicle) nanoparticles are shown to have higher ice recrystallization inhibition activity compared to smaller (sphere) particles, whereas ice nucleation activity was not found for any material. This shows that assembly into larger structures can increase IRI activity and that increasing the “size” of an IRI does not always lead to ice nucleation. This nanoparticle approach offers a platform toward ice-controlling soft materials and insight into how IRI activity scales with molecular size of additives.

Topology Reliable LCST‐Type Behavior of ABA Triblock Polymer and Influence on Water Condensation and Crystallization

As a kind of smart material, thermoresponsive hydrogels are widely investigated and applied in many fields. Due to the limitation of the freezing temperature of the water, it is a challenge to further broaden their sol-gel transition temperature (Tgel ) range, especially below 0°C. Herein, the lower critical solution temperature type of amphiphilic ABA triblock copolymers, synthesized via two-step reversible addition-fragmentation chain transfer (RAFT) polymerization is demonstrated. The hydrophilic A-block and the hydrophobic B-block are composed of poly(N,N-dimethylacrylamide) (PDMAA) and poly(diacetone acrylamide) (PDAAM), respectively. The degree of polymerization (DP) of both A-block and B-block shows a significant influence on the Tgel of triblock copolymer dispersion. By changing the length of these two blocks or physically blending these copolymers dispersions, the Tgel can be well adjusted in a temperature range from 45 to -10°C. Moreover, When the Tgel is higher than 4°C, the triblock copolymer coatings show a good anti-fogging property. And when the Tgel is around or lower than the freezing temperature of the water, aqueous dispersions of the triblock copolymer have an ice recrystallization inhibition activity, resulting in the decrease of average maximum grain size (MLGS) of ice crystal.