Ice Recrystallization Inhibition Research Articles

Amyloid protein fibrils show enhanced ice recrystallization inhibition activity when serve as pickering emulsion stabilizer

Ice recrystallization adversely affects the quality of frozen foods. Therefore, food-grade materials with ice recrystallization inhibition (IRI) activity are attracting growing attention. Our previous study has found IRI activity of amyloid protein fibrils (APFs); however, as with most other food-grade IRI active materials, the IRI activity of APFs is moderate and needed to be enhanced. Here, we report that IRI activity is greatly enhanced when APFs serve as Pickering emulsion stabilizer. Adsorption of APFs to the oil-water interface resulted in the formation of microsized Pickering emulsion droplets. The IRI activity resulted from the adsorbed APFs rather than steric hindrance of emulsion droplets because self-induced emulsion droplets without APFs on the surface had no IRI activity. The oil core chemistry does not affect the IRI activity of adsorbed APFs. A structure−activity relationship analysis revealed that IRI activity of adsorbed APFs increases with larger particle size and emulsion droplet number (total number of droplets per unit volume). The enhancement of IRI activity caused by oil-water interface adsorption is reasonably ascribed to enlarged size, crowding of APFs on the surface of emulsion droplets, and enhanced ice adsorption of APFs. Compared with free APFs, adsorbed APFs were more IRI active at higher annealing temperatures and higher unfrozen water contents. Given the amphiphilic nature of most IRI-active materials, the findings of this study shed light on the application of IRI-active materials as emulsifiers to improve the quality of frozen food containing emulsions, such as ice cream and frozen meat paste.

A novel automated protocol for ice crystal segmentation analysis using Cellpose and Fiji

Accurate measurement of ice crystal size is an essential step in quantitative ice recrystallization inhibition (IRI) analysis using the sucrose sandwiching assay (SSA) and splat assay (SA). Here, we introduce a novel method of measuring ice crystal size and shape using Fiji and Cellpose, an anatomical segmentation algorithm, to address the time-consuming and limited number of ice particle determination associated with the mean largest grain size measurement. This new automated approach, displaying rapid segmentation of ∼70 s per image, measures every ice crystal in an image field of view, consequently reducing bias introduced by subjectively selecting the largest crystals in an image. Consistent in determining a diverse set of crystal sizes and shapes, this method allows for the evaluation of ice crystals using Feret's diameter, a parameter that better accounts for irregular particle shape. This method provides new outputs such as standard deviation, particle size distributions of a population of ice crystals, and circularity to characterize and further provide insight into an analyte's IRI ability. Applicable to the SSA, the “shape descriptor” measurement can be used to quantify ice binding. This work presents a novel and accurate approach for ice crystal quantitative analysis.

Depletion interaction may reduce ice recrystallization inhibition activity of cellulose nanocrystals (CNCs) at high concentrations

In ice cream, food hydrocolloids are commonly used as stabilizers for controlling ice recrystallization. Recent studies suggest that some stabilizers might bind to ice crystal surfaces and inhibit recrystallization. However, this ice-binding mechanism is challenged by the fact that stabilizers at high concentrations sometimes have reduced activity or even accelerate ice recrystallization. In this work, we determined the ice recrystallization inhibition (IRI) activity of cellulose nanocrystals (CNCs) at different concentrations in the 3.0% sucrose solution by the splat assay and in the 25.0% and 40.0% sucrose solutions by the sandwich assay. Reduced activity at high CNCs concentrations was observed in the sandwich assay. It was not linked to the phase separation or liquid crystal phase formation of CNCs but was caused by the accretion of nearby ice crystals and the forming of large ice crystals with irregular morphology and high aspect ratios. A new explanation based on the depletion-interaction-induced accretion of ice crystals was proposed. Our findings might resolve the contradiction between the ice-binding mechanism and the stabilizer concentration effect and add more support to the interfacial mechanism of ice recrystallization inhibition. They also pointed out the importance of selecting suitable stabilizer concentrations for screening IRI active materials and manufacturing frozen desserts.

Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells

BackgroundA medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at –196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.MethodAs biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at –196 °C for 7 days. Then, cell recovery was assayed.ResultsWe found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.ConclusionsPEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.Graphical

Ice Nucleation Promotion Impact on the Ice Recrystallization Inhibition Activity of Polyols.

Heterogeneous ice nucleation occurs vis-à-vis nucleating agents already present in solution yet can occur within a rather broad range of temperatures (0 to ca. -38 °C). Controlling this temperature and the subsequent growth of resulting ice crystals is crucial for the survival of biological organisms (certain insects, fish, and plants that endure subzero temperatures), as well as in the context of medical cryopreservation and food science. In these environments, uncontrolled crystal shape and size can rupture the cell membrane causing irreversible and catastrophic damage. Antifreeze (AF) proteins and synthetic AF analogs address this issue to restrict crystal growth and to shape ice crystals. Yet, if the nucleation temperature is not controlled and occurs in a lower temperature range, nascent ice crystals will have grown to a significantly larger size before the AF agents can be active on their surface to halt or slow the Ostwald ripening process during recrystallization. At a higher nucleation temperature, diffusion of AF macromolecules is enhanced, and dynamic crystal shaping can start earlier, producing smaller crystals overall. While antifreeze proteins, the inspiration for these synthetic analogs, are always applied in a salt buffer aqueous environment (most typically phosphate-buffered saline (PBS) buffer), the heterogeneous nucleation events are stochastic and occur within a wide temperature range. Silver iodide (AgI), however, is a highly effective ice nucleation promoter as its crystal lattice structure is a 98% lattice match to the basal plane of hexagonal ice (Ih) crystals acting as a template for water molecule orientation and decreasing the interfacial free energy. Here, we expose the advantage of purposely seeding such nascent ice crystals with AgI at a defined and higher temperature (-7 °C) in ultrapure water (UPW) such that nucleation can only come from AgI (and also in AgI/PBS), resulting in the most potent synthetic IRI observed to date (at concentrations as low as 0.001 mg·mL-1).

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50μg dry TMT per channel was used to label 6-12μg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

Mechanistic insights into the improvement of yeast viability by adding short-clustered maltodextrin during long-term frozen storage

The formation and growth of ice crystals are widely acknowledged to cause damage to different substances and we found short-clustered maltodextrin (SCMD) could alleviate the quality deterioration of frozen dough in previous research. But how SCMD interacts with dough components such as yeast is poorly understood. Here, we analyze the ice-recrystallization-inhibition (IRI) activity of SCMD and provide mechanistic insights into the improvement of yeast viability by adding SCMD during long-term frozen storage. SCMD had a higher IRI activity compared with DE2 maltodextrin, trehalose and glycerin at the same dosage. Through untargeted metabolomics analysis, SCMD addition proved to be enhancing lipid metabolism, regulating amino acid metabolism and energy metabolism of yeast, making yeast cells possess more similarities to the positive control with minimal freeze injury. In contrast, no significant protective effects were observed with low-concentration trehalose and glycerin. Our reports reveal the cryoprotection mechanism of SCMD from the perspective of the cellular metabolism of yeast, providing the foothold for further exploration of SCMD as a safe and efficient cryoprotectant in different fields. • Short-clustered maltodextrin (SCMD) could change ice-crystal shapes. • SCMD had a higher ice-recrystallization-inhibition (IRI) activity than trehalose and glycerin at the same dosage. • SCMD addition could improve yeast viability and fermentation ability during long-term frozen storage. • SCMD addition enhanced lipid metabolism, regulated amino acid metabolism and energy metabolism of yeast. • SCMD cryoprotection mechanism was proposed.

Ice recrystallization inhibition mechanism of zwitterionic poly(carboxybetaine methacrylate).

Understanding the ice recrystallization inhibition (IRI) mechanism is of fundamental importance for the rational design of novel antifreeze protein mimetics and reducing IR-related damage. In this communication, using quantitive experimental methods and molecular dynamics simulations we demonstrate that zwitterionic poly(carboxybetaine methacrylate) (PCBMA) can serve as a novel IRI-active substance. This work unravels the atomic-level details of the IRI mechanism of zwitterionic antifreeze protein mimetics and provides insight into the development of next-generation antifreeze protein mimetics.

Investigation into antifreeze performances of natural amino acids for novel CPA development.

Cryopreservation can prolong the viability of cells and help meet the demand for biosamples of high medical value. Cryoprotectants (CPAs) can mitigate unavoidable cell cryoinjury caused by the formation and growth of ice crystals during freeze-thaw cycles. Therefore, the development of efficient and biocompatible CPAs has attracted extensive attention. In this work, the antifreeze performances of 18 water-soluble natural amino acids were systematically investigated by wet experiments and molecular dynamics simulations to explore their potentials as CPAs. Phenylalanine has an amphipathic structure with excellent ice recrystallization inhibition (IRI) activity, and methionine exhibits optimal depressing freezing point ability. Combining phenylalanine or methionine with osmolyte (proline) as CPAs can maintain normal morphology of sheep red blood cells (RBCs), and the recovery of sheep RBCs reaching 85 and 71%, respectively, while only 32% in the 20% (w/v) glycerol solution. This study demonstrates the potential of amino acid-based CPAs and reveals the synergistic effect of IRI activity and osmotic pressure regulation in cryopreservation.

Quantified instant conjugation of peptides on a nanogold surface for tunable ice recrystallization inhibition.

The adverse effects of recrystallization limit the application of cryopreservation in many fields. Peptide-based materials play an essential role in the antifreezing area because of their excellent biocompatibility and abundant ice-binding sites. Peptide-gold nanoparticle conjugates can effectively reduce time and material costs through metal-thiol interactions, but controlled modification remains an outstanding issue, which makes it difficult to elucidate the antifreezing effects of antifreeze peptides at different densities and lengths. In this study, we developed an instant peptide capping on gold nanoparticles with butanol-assisted dehydration and provided a controllable quantitative coupling within a certain range. This chemical dehydration makes it possible to fabricate peptide-gold nanoparticle conjugates in large batches at minute levels. Based on this, the influence of the peptide density and sequence length on the antifreezing behaviors of the conjugates was investigated. The results evidenced that the antifreezing property of the flexible peptide conjugated on a rigid core is related to both the density and length of the peptide. In a certain range, the density is proportional to the antifreeze, while the length is negatively correlated with it. We proposed a rapidly controllable method for synthesizing peptide-gold nanoparticle conjugates, which may provide a universal approach for the development of subsequent recrystallization-inhibiting materials.

Microcurvature Controllable Metal-Organic Framework Nanoagents Capable of Ice-Lattice Matching for Cellular Cryopreservation.

Ice-binding proteins (IBPs) produced by psychrophilic organisms to adapt for the survival of psychrophiles in subzero conditions have received illustrious interest as a cryopreservation agent required for cells and tissues to completely recover after freezing/thawing. Depressing water-freezing point and avoiding ice-crystal growth affect their activities which are closely related to the presence of ice crystal well-matched binding moiety. The interaction of IBPs with ice and water is critical in enhancing their freeze avoidance against cell or tissue damage. Metal-organic frameworks (MOFs) with a controllable lattice at the molecular level and a size at the nanometer scale can offer periodically ordered ice-binding sites by modifying organic linkers and controlling microcurvature at the ice surface. Herein, zirconium (Zr)-based MOF-801 nanoparticles (NPs) with good biocompatibility were used as a cryoprotectant that is well dispersed and colloidal-stable in an aqueous solution. The MOF NP size was precisely controlled, and 10, 35, 100, and 250 nm NPs were prepared. The specific IBPs-mimicking pendants (valine and threonine) were simply introduced into the MOF NP-surface through the acrylate-based functionalization to endow with hydrophilic and hydrophobic dualities. When small-sized MOF-801 NPs were attached to ice, they confined ice growth in high curvature between the adsorption sites because of the decreased radius of the convex area of the growth region, leading to highly enhanced ice recrystallization inhibition (IRI). Surface-functionalized MOF NPs could increase the number of anchored clathrate water molecules with hydrophilic/hydrophobic balance of the ice-binding moiety, effectively inhibiting ice growth. The MOF-801 NPs were biocompatible with various cell lines regardless of concentration or NP surface-functionalization, whereas the smaller-sized surface-functionalized NPs showed a good cell recovery rate after freezing/thawing by induction of IRI. This study provides a strategy for the fabrication of low-cost, high-volume antifreeze nanoagents that can extend useful applications to organ transplantation, cord blood storage, and vaccines/drugs.

Investigation on the quality regulating mechanism of antifreeze peptides on frozen surimi: From macro to micro

Freeze denaturation of protein caused by ice crystals is the main motivation for the quality deterioration of surimi during circulation and storage. This investigation aimed to cryoprotect surimi by adding antifreeze peptides from Takifugu obscurus skin (TsAFP) which can inhibit ice recrystallization, and to elucidate regulating mechanism. The comprehensive results showed that 4% TsAFP, half dosage of commercial cryoprotectant, had good cryoprotection on surimi by reducing the moisture variation and maintaining protein solubility of surimi at macro level, as well as inhibiting the degeneration and structure changes of myofibrillar proteins at micro level. Meanwhile, TsAFP could directly bind to the structural cavity of myosin, inhibit protein freezing-induced oxidation, maintain the spatial structure of myosin and water retention ability to preserve the surimi quality. This study helped better comprehend the protective mechanisms of antifreeze peptides in frozen surimi and was expected to provide a promising cryoprotectant for low-sweetness and low-calorie surimi.

Inhibiting ice recrystallization by amyloid protein fibrils

Ice recrystallization is harmful to the quality of frozen foods and the cryopreservation of cells and biological tissues, requiring biocompatible materials with ice recrystallization inhibition (IRI) activity. Emerging studies have associated IRI activity with amphiphilic structures. We propose amphiphilic amyloid protein fibrils (APFs) may be IRI-active. APFs were prepared from whey protein isolate (WPI) in water (W-APFs) and in trifluoroethanol (TFE-APFs). W-APFs and TFE-APFs were more IRI-active than WPI over a concentration range of 2.5–10.0 mg/mL. Both APFs showed stronger IRI activity at pH 3.0 than at pH 5.0, 7.0, and 10.0, which was ascribed to the effect of water dispersibility and fibril length. The reduced IRI activity of the two APFs with increasing NaCl content was caused by fibril aggregation. Ice binding by APFs was absent or very weak. Ordered water was observed for the two APFs, which might be essential for IRI activity. Our findings may lead to the use of APFs as novel ice recrystallization inhibitors.

Probing interactions between small-molecule ice recrystallization inhibitors (IRIs) and water molecules using 1H NMR

Probing interactions between small-molecule ice recrystallization inhibitors (IRIs) and water molecules using 1H NMR

Isolation and characterization of wheat ice recrystallisation inhibition gene promoter involved in low temperature and methyl jasmonateresponses.

It is well known that plant growth, development, survival and geographical distribution are constrained by extreme climatic conditions, especially extreme low temperature. Under cold stress, cold-inducible promoters were identified as important molecular switches to transcriptionally regulate the initiation of genes associated with cold acclimation processes and enhance the adaptability of plants to cold stimulation. Wheat (Triticum aestivum L.) is one of the most dominating food crops in the world, andwheat crops are generally overwintering with strong cold resistance. Our previous study already proved that heterologous expression of wheat ice recrystallization inhibition (IRI) genes enhanced freezing tolerance in tobacco. However, the upstream regulatory mechanisms of TaIRI are ambiguous. In this study, the space-time specific expression of TaIRI genes in wheat was analyzed by quantitative real-time PCR (qRT-PCR), and results showed that the expression of TaIRI in all tissues was cold-induced and accelerate by exogenous methyl jasmonate (MeJA). Three promoters of TaIRI genes were isolated from wheat genome, and various 5'-deletion fragments of TaIRIp were integrated into β-glucuronidase (GUS) within vector pCAMBIA1301. The promoteractivity of TaIRI geneswas determined through transient expression system of tobacco and stable expression of Arabidopsis thaliana. Results revealed that the GUS activity were significantly strengthened by cold and MeJA treatments. This study will provide insights into elucidating the transcription-regulatory mechanism of IRI proteins responding to low temperature.

The gelatin-based liquid marbles for cell cryopreservation.

As an alternative and a straightforward cryopreservation biotechnological tool, liquid marble provides a promising cryopreservation approach. Currently, effective cell preservation mainly based on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). As state-of-the-art cryoprotectant (CPA), DMSO, has intrinsic toxicity, which is the bottleneck of its widespread application. The complex compositions of FBS have the potential risks of pathogenic microorganism contamination. However, efficient cell cryopreservation using liquid marbles, a platform independent of DMSO and FBS, has not been well investigated yet. Herein, we explore the cryoprotection role of liquid marbles based on gelatin solution. Gelatin has a superior biocompatibility, which DMSO is incomparable. During a freeze-thaw cycle, gelatin produces negligible osmotic pressure, and has high ice recrystallization inhibition (IRI) activity to induce the formation of smaller and smooth ice crystals. Moreover, the specific structure of liquid marble also provides favorable supports for cell survival. The cryopreservation efficiency of mouse fibroblasts cells L929 via the gelatin-based liquid marble was as high as 90%, and the recovered cells could maintain their normal functionalities. This work opens a new window of opportunity for non-toxic and efficient cryopreservation of liquid marbles without the need of DMSO and FBS addition.

Thermal hysteresis and ice recrystallization inhibition activities of antifreeze protein-poly(vinyl alcohol) conjugates

Thermal hysteresis and ice recrystallization inhibition activities of antifreeze protein-poly(vinyl alcohol) conjugates

Experimental proof of ice recrystallization inhibition on the C. elegans model based on a scalable warming approach by means of high intensity focused ultrasound

Experimental proof of ice recrystallization inhibition on the C. elegans model based on a scalable warming approach by means of high intensity focused ultrasound

Small molecule ice recrystallization inhibitors dramatically improve the cryopreservation of cellular therapy products

Small molecule ice recrystallization inhibitors dramatically improve the cryopreservation of cellular therapy products