Inhibit Ice Crystal Growth Research Articles

Hydrogel encapsulation facilitates a low-concentration cryoprotectant for cryopreservation of mouse testicular tissue

Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction.

Higher molecular weight pectin inhibits ice crystal growth and its effect on the microstructural and physical properties of pectin cryogels

Understanding the formation of ice crystals is essential for tailoring the microstructure and physical properties of cryogels. This study investigated the effects and mechanisms of pectin molecular weight (Mw) on impacting ice crystal formation. Pectin fractions various Mw (10.13–212.20 kDa) were prepared by hydrothermal method. The solution of high Mw pectin fractions exhibited higher contact angle, lower water freedom, and stronger adsorption of water molecules. The splat experiment and molecular dynamic (MD) results confirmed that higher Mw pectin have stronger ice crystal growth inhibition activity than lower Mw pectin. Furthermore, the pore size distribution of the cryogel increased from 98–203 μm to 105–267 μm as the molecular weight decreased from 212.2 kDa to 121.0 kDa. Additionally, in the higher Mw pectin cryogel, stronger mechanical strength was observed. These findings suggested that changing the molecular weight of pectin has the potential to regulate the ice crystal growth, microstructure and physical properties of frozen products.

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.

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.

Ice Recrystallization Inhibition Activity of Soy Protein Hydrolysates.

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. The objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1-F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57-29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4-14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

Identification of Novel Antifreeze Peptides from Takifugu obscurus Skin and Molecular Mechanism in Inhibiting Ice Crystal Growth.

Foodborne hydrolyzed antifreeze peptides have been widely used in the food industry and the biomedical field. However, the components of hydrolyzed peptides are complex and the molecular mechanism remains unclear. This study focused on identification and mechanism analysis of novel antifreeze peptides from Takifugu obscurus skin by traditional methods and computer-assisted techniques. Results showed that three peptides (EGPRAGGAPG, GDAGPSGPAGPTG, and GEAGPAGPAG) possessed cryoprotection via reducing the freezing point and inhibiting ice crystal growth. Molecular docking confirmed that the cryoprotective property was related to peptide structure, especially α-helix, and hydrogen bond sites. Moreover, the antifreeze peptides were double-faces, which controlled ice crystals while affecting the arrangement of surrounding water molecules, thus exhibiting a strong antifreeze activity. This investigation deepens the comprehension of the mechanism of antifreeze peptides at molecular scale, and the novel efficient antifreeze peptides can be developed in antifreeze materials design and applied in food industry.

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

Identification of antifreeze peptides in shrimp byproducts autolysate using peptidomics and bioinformatics

In the present study, a novel method based on peptidomics and bioinformatic was applied to identification and characterization of antifreeze peptides (AFPs) from shrimp byproducts autolysate (SBPA). According to the results of in silico prediction and high peptide structural inflexibility, DEYEESGPGIVH and EQICINFCNEK were picked as potential AFP-1 and AFP-2, respectively. The outcomes of DSC determination indicated that TH of synthesized AFP-1 and AFP-2 (10 mg/mL) were 1.37 °C and 1.57 °C, respectively. Besides, 0.1 %-3 % AFPs showed significant cryoprotection in shrimp muscle after 3 and 6 freeze–thaw cycles, evidenced by higher SSP content, Ca2+-ATPase activity, sulfhydryl content and lower surface hydrophobicity than control; while the higher concentration resulted in better protection against freeze induced denaturation. Both AFP-1&2 showed favorable hydrogen bonding affinity which facilitated ice binding and ice crystal growth inhibition. This work could provide new ideals for identification and characterization of AFPs.

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.

The inhibitory mechanism of l-lysine hydrochloride on the ice crystals growth by molecular dynamics

Understanding the inhibitory mechanism of cryoprotective agent on ice growth is essential for preventing the cryoinjury of vivo biological tissue and for the bioactive protection of protein medicine in cryopreservation processes. The crystallization growth process of (0 2 0) crystal face of orthorhombic ice in l-lysine hydrochloride aqueous solutions was researched by molecular dynamic. The results indicated that l-lysine ion has a competitive adsorption on the lattice sites of ice interface with water molecule. The binding carboxyl (COO−) and flexible l-lysine chain can disrupt the periodic continuous growth of ice. The free l-lysine ion and chlorine anion (Cl−) inhibit the diffusion of water molecules which makes ice crystals difficult to grow. This final results proved that l-lysine ion can inhibit ice crystal growth in both aqueous and crystalline phases.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ± 0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 μM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

Ice-Binding Protein from Shewanella frigidimarinas Inhibits Ice Crystal Growth in Highly Alkaline Solutions.

The ability of a natural ice-binding protein from Shewanella frigidimarina (SfIBP) to inhibit ice crystal growth in highly alkaline solutions with increasing pH and ionic strength was investigated in this work. The purity of isolated SfIBP was first confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography with an ultraviolet detector (SEC-UV). Protein stability was evaluated in the alkaline solutions using circular dichroism spectroscopy, SEC-UV, and SDS-PAGE. SfIBP ice recrystallization inhibition (IRI) activity, a measure of ice crystal growth inhibition, was assessed using a modified splat assay. Statistical analysis of results substantiated that, despite partial denaturation and misfolding, SfIBP limited ice crystal growth in alkaline solutions (pH ≤ 12.7) with ionic strength I ≤ 0.05 mol/L, but did not exhibit IRI activity in alkaline solutions where pH ≥ 13.2 and I ≥ 0.16 mol/L. IRI activity of SfIBP in solutions with pH ≤ 12.7 and I ≤ 0.05 mol/L demonstrated up to ≈ 66% reduction in ice crystal size compared to neat solutions.

Role of synthetic antifreeze agents in catalyzing ice nucleation

Nature endows antifreeze (glyco)proteins (AF(G)Ps) with the excellent capability of inhibiting ice crystal growth. Recent years have also witnessed the emergence of many potent AF(G)P mimics such as poly (vinyl alcohol) (PVA). As researchers are revealing the molecular mechanisms of inhibiting ice crystal growth by AF(G)Ps and their synthetic substitutes, there remains no agreement about their effect on ice nucleation. In this study, we report the observation of ice nucleation catalyzed by PVA of different polymerization degrees using a freeze-on-a-chip platform which allows the monitoring of freezing and melting events over hundreds of monodisperse, picoliter-sized aqueous droplets. Aqueous droplets made of 1 mg/ml PVA solution exhibit a median freezing temperature of around −36 °C, two degrees higher than the observed homogeneous nucleation temperature of water. The findings in our study bring useful insights into the different roles of synthetic antifreeze agents in controlling ice formation.

Molecular Dynamics Simulation of Ice Crystal Growth Inhibition by Hexadecyl-trimethyl-ammonium Bromide.

Recent experiments have found hexadecyl-trimethyl-ammonium bromide (CTAB) to have superior ice nucleation inhibition properties [ J. Phys. Chem. B 121, 6580]. The mechanism of how the inhibition takes place remains unclear. Therefore, molecular dynamics was used to simulate ice crystallization of a water/CTAB/ice system. The ice crystallization rate for a pure water system was compared for the basal [0001], first prism [101̅0], and secondary prism plane [112̅0], where the basal plane grew the slowest followed by the first prism plane. When CTAB was added to the ice-liquid water system, crystallization was clearly impeded. Even when ice starts growing away from the CTAB molecule, the hydrophilic head would at some point protrude and get caught in the water/ice interface. Once the head of the CTAB was encapsulated in the advancing interface, the hydrophobic body would wriggle around and disrupt the formation of hydrogen bond networks that are essential for ice growth. When the interface clears the length of the body of the CTAB molecule, ice crystallization resumes at its normal pace. In summary, the inhibition of ice growth is a combination of the hydrophilic head acting as an anchor and the dynamic motion of the hydrophobic tail hindering stable hydrogen bonding for ice growth.

Ice crystal growth inhibition by peptides from fish gelatin hydrolysate

Ice crystal growth inhibition in an ice cream mix matrix and in sucrose solutions by peptides derived from alcalase (also known as subtilisin) hydrolyzed fish gelatin was investigated. Hydrolysis of fish gelatin at an optimum hydrolysis condition (i.e. 20% w/w gelatin solution treated with Alcalase at an enzyme-to-substrate ratio of 0.176 Anson units/g gelatin at pH 9.0 for 25 min at 45 °C) released peptides with maximum ice crystal growth inhibition activity. Fractionation of gelatin hydrolysate using size exclusion and ion exchange chromatography resulted in isolation of a cationic peptide fraction containing two prominent peptides having 1850.82 Da and 2036.88 Da molecular masses. This cationic fraction had the highest ice crystal growth inhibition activity. The amino acid sequences of these peptides showed no sequence similarity other than that they both contained –GTPG- and –GPP(OH)G-motifs and 3 to 5 hydroxyl containing amino acid residues. The results of this study supported the hypothesis that short collagen/gelatin polypeptides in the molecular mass range of 1000–2500 Da, regardless of their source, would have the ability to inhibit ice crystal growth in frozen systems. The results also suggested that the mechanism of ice crystal growth inhibition by gelatin peptides might involve three steps, namely, initial nonspecific electrostatic interaction of cationic peptides with the negatively charged ice surface, followed by structural realignment to optimally hydrogen bond with the oxygen-oxygen lattice on the ice surface, and stabilization of the electrostatic and hydrogen bonding in the peptide – ice crystal complex by a partial nonpolar environment created by neighboring hydrophobic residues of the peptide.

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

Stabilising frozen dairy mousses by low molecular weight gelatin peptides

The effect of low molecular weight gelatin peptides on the shrinkage of thawed mousses was investigated. Changes promoted by freezing-thawing processes were evaluated through texture and volume measurements and through X-ray analysis of the bubble distribution. Freezing provoked collapse of the standard reference recipe mousse and of the reference recipe mousse with 2% milk powder added, but samples containing 2% gelatin peptides showed no shrinkage. The bubble size and bubble number distribution of the different mousses were measured based on high-resolution X-ray tomography. Results indicated that the volume losses experienced by the controls were almost entirely caused by the disappearance of air bubbles having a diameter smaller than 50 μm. Hence, this fraction of over-pressurised air bubbles is extra stabilised by the matrix due to the additional presence of gelatin peptides. Moreover, gelatin peptides were found to inhibit ice crystal growth, which resulted in smaller ice crystals that are believed to be less destructive to the microstructure of the freeze-thawed mousses.

Antifreeze proteins enable plants to survive in freezing conditions.

Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules.

Chemical synthesis of a masked analogue of the fish antifreeze potentiating protein (AFPP)

A recently identified Antarctic fish protein termed antifreeze potentiating protein (AFPP) is thought to act as an adjunct to the previously characterised antifreeze glycoproteins (AFGPs), the two acting together to inhibit ice crystal growth in vivo. Elucidating the functional properties of the new AFPP requires access to large amounts of pure product, but the paucity of natural material necessitates alternative approaches. We therefore embarked on the total chemical synthesis of the AFPP, through a convergent ligation strategy. After many challenges, mostly due to the solubility issues of the peptide fragments, and several revisions of the original synthetic strategy, we have successfully synthesized a masked analogue of AFPP. The key to the successful synthesis was the use of a solubilising tag attached through a hydrolysable linker.