Recrystallization Inhibition Activity Research Articles

A Close Cognition of Charged Poly(l-methionine) Derivatives for Antifreeze.

Ice formation poses a significant challenge across various fields, from industrial processes to biological preservation. Developing antifreeze agents and recognizing the antifreeze mechanism have gained considerable attention. Herein, a series of poly(l-methionine) derivatives, poly(S-carboxymethyl-l-methionine sulfonium) (PMetA), poly(S-methyl-l-methionine sulfonium chloride) (PMetM), and poly(S-carbamidomethyl-l-methionine sulfonium chloride) (PMetAM), with carboxyl, methyl, and acetamide groups, respectively, are synthesized and investigated for antifreeze. The relationship between the polymer structure and the ice recrystallization inhibition (IRI) activity is examined, suggesting that zwitterionic PMetA shows the highest IRI activity, about 27.0 ± 3.9% at 10 mg mL-1 relative to that of water. Results of low-field nuclear magnetic resonance and differential scanning calorimetry indicate that the IRI activity is associated with the activation energy for hydrogen bond breakage. PMetA exhibits acceptable cytocompatibility at 10.0 mg mL-1 and a good cryoprotective efficiency. This finding provides a valuable insight into the antifreeze mechanism, contributing to the development of potent cryoprotectants.

Effect of pectin with different esterification degree on the freeze-thaw stability of gluten protein: Structures, functional properties, and Cryoprotective mechanism

The effect of degree of esterification (DE) on the cryoprotective function of pectin against the frozen deterioration of gluten protein is still unclear. Here, NMR, HPSEC-MALLS, HPGPC and optical microscrope were applied to determine the structure and ice recrystallization inhibition (IRI) activity of pectin with varying DE. The effect on structure, physicochemical properties and molecular interaction of gluten supplemented with pectin were investigated. Results suggested that low-ester pectin (LEP) exhibited higher IRI activity than that of high-ester pectin (HEP), and reduced the damage of gluten during freezing storage. Moreover, SEM and low field-NMR revealed that LEP effectively helped to maintain the network structure and inhibited the water migration of gluten. Besides, molecular docking results suggested higher affinity between LEP and gluten protein than that of HEP. These results illustrated better cryoprotective effects of LEP than that of HEP through pathways including IRI activity, water migration, and possible molecular interaction.

Soy protein processing impacts the ice recrystallization inhibition activity of protein hydrolysates

AbstractSoy protein hydrolysates have demonstrated moderate ice recrystallization inhibition (IRI) activity, but the properties of the unhydrolyzed protein contributing to this activity are not well known. The objective of this research was to identify the main protein processing factors important to the varying IRI activities observed. Three possible modification reactions were applied to soy protein isolate (SPI): the Maillard reaction that can occur in soy flour before protein isolation, heat denaturation of the fully defatted protein, and heat denaturation of the protein with the presence of residual lipid, mainly polar phospholipids. These modified proteins were hydrolyzed by Alcalase protease (for 2 min) to produce hydrolysates, which were characterized by HPLC and FTIR to investigate how molecular weight (MW) and changes in secondary structure relate to IRI activity. A multilinear regression with the parameters of modification type, surface hydrophobicity, secondary structure profile, and average MW of the hydrolysates was used to investigate their roles in reducing ice crystal size. It was discovered that polar lipids present in the soy hydrolysate samples and MW were the only significant factors (p < 0.05) for IRI activity of SPI hydrolysates which had an ice crystal size reduction from 22.0% to 36.7%. This study demonstrates for the first time a protein hydrolysate‐phospholipid interaction can produce IRI active molecules.

Data-driven discovery of potent small molecule ice recrystallisation inhibitors

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain.

Effect of the Hofmeister series anion salts on the ice recrystallization inhibition activity of tamarind seed polysaccharide

Tamarind seed polysaccharide (TSP) has been identified as an ice recrystallization inhibition (IRI)-active polysaccharide with potential applications in food cryoprotection. In this study, the IRI performance of TSP was investigated using the splat assay in the presence of different salts containing Hofmeister series anions (Na2SO4, NaH2PO4, NaCl, and NaNO3). The results showed that the IRI activity of TSP in different salt solutions depended on the type of ion present, in agreement with the reverse Hofmeister anion sequence: NO3− > Cl− > H2PO4− > SO42−. It was discovered that the Hofmeister series anion salts modulate the IRI activity of TSP by inducing changes in the aggregation and hydration properties of polysaccharides. In particular, TSP exhibited greater IRI activity when characterized by a lower aggregation and higher hydration degree in the presence of chaotropic ions. These findings highlight the influence of additives present in cryopreservation media and contribute to a fundamental understanding of the mechanism underlying the differential influence on the performance of IRI-active polysaccharides.

Ice recrystallization inhibition activity of pulse protein hydrolysates after immobilized metal affinity separation

Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.

Development of Macromolecular Cryoprotectants for Cryopreservation of Cells.

Cryopreservation is a common way for long-term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane-impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo-injury and are easily removed post-thaw, are promising candidates with biocompatibility and feasibility. Water-soluble hydroxyl-containing polymers such as poly(vinyl alcohol) and polyol-based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio-inspired (glyco)polypeptides can significantly increase post-thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells.

Furcellaran: Impact of Concentration, Rheological Properties, and Structure on Ice Recrystallization Inhibition Activity.

Recrystallization is considered the main damaging mechanism during the frozen storage of biologic materials. In this study, furcellaran, a polysaccharide related to κ-carrageenan, was studied for its concentration-dependent effect on ice crystal growth and recrystallization. The structure and sulfate content of the utilized furcellaran was analyzed by 1H nuclear magnetic resonance spectroscopy, ion chromatography, and high-performance size-exclusion chromatography. Additionally, the rheological properties of furcellaran solutions were investigated. Our findings demonstrate that furcellaran inhibits ice growth as effectively as κ-carrageenan. Furthermore, the rheological properties change with increasing furcellaran concentration, resulting in a gel-like consistency at 5 g/L, which coincides with decreased recrystallization inhibition activity and larger crystals. This suggests that gel formation or a gel-like consistency has to be avoided for optimal recrystallization inhibition activity.

Chickpea peptide as a plant-based cryoprotectant in frozen dough: Insight into the water states, gluten structures, and storage stabilities

The potentials of pulse peptides as cryoprotectants remain less exploited than other well-known applications, e.g., nutrient supplements, stabilizers, antioxidants etc. Thus, for the first time, this study screened a number of widely-used pulse peptides (pea, chickpea, lentil, mung bean, and white kidney bean) by evaluating their ice recrystallization inhibition (IRI) and thermal hysteresis (TH) activities. Then, chickpea peptide (CP) with the most prominent anti-freezing performance was selected for systematical evaluation of its cryoprotective effects on frozen dough in terms of water state, gluten structure, and storage stability. CP reduced the content of freezable water to 60% and unified the water distributions of frozen dough in a dose-dependent manner (0–6 g peptide per 100 g wheat flour). Freezing-induced changes on gluten disulfide bonds, secondary structures, and dough microstructures were also restrained. Accordingly, dumpling wrappers prepared from CP-added frozen dough manifested the reduced cracking rates (31%–5%), increased toughness (68%–79%), extensibility (82%–92%), and cooking stabilities. CP was rich in basic amino acids (arginine, lysine, and histidine), accounting for 27% of total amino acids, which were responsible for its outstanding cryoprotective activity. Chickpea peptide might therefore be a good candidate for development of high-performance dough cryoprotectants.

The ice recrystallization inhibition activity of wheat glutenin hydrolysates and effect of salt on their activity

This study aimed to investigate the antifreeze activity of wheat glutenin hydrolysates produced by Alcalase and trypsin at varying degrees of hydrolysis. The influence of salt concentrations in two dispersing media, phosphate-buffered saline (PBS) and NaCl solution, and the types of cations, NaCl and CaCl2, on hydrolysates was further analyzed. The results revealed a consistently higher ice recrystallization inhibition (IRI) activity for all hydrolysates dispersed in 0.1 x PBS than in 1 x PBS. Interestingly, glutenin hydrolysates exhibited better IRI activity in CaCl2 than in NaCl solution under the same ionic strength. Furthermore, trypsin-derived hydrolysates (with a molar mass about 10 kDa) consistently had better IRI activity than those produced by Alcalase (with a molar mass less than 1 kDa), regardless of the dispersing media. The observed increase in IRI activity was associated with alterations in the secondary structure, characterized by a higher α-helix and lower β-turns content. This suggests that structural rigidity of the molecules plays a significant role in enhancing the antifreeze activity of peptides. Additionally, the scores plot of the partial least-squares discriminant analysis model indicates that salt concentration had a more significant impact than salt type on the IRI activity of glutenin hydrolysates. In conclusion, our findings provide insights into the factors influencing the IRI activity of protein hydrolysates.

Effects of Flammulina velutipes polysaccharide with ice recrystallization inhibition activity on the quality of beef patties during freeze-thaw cycles: An emphasis on water status and distribution

The antifreeze activity of Flammulina velutipes polysaccharide (FVP) autoclave-extracted with dilute alkaline and effects of FVP on moisture status, size of ice crystals, physical and chemical characteristics of beef patties during repeated freeze-thaw (F-T) cycles were investigated. Results showed that FVP exhibited ice recrystallization inhibition activity and was able to alter the onset freezing/melting temperature of beef patties. 0.01% FVP significantly alleviated (P < 0.05) the decrement in water holding capacity by inhibiting water migration, restraining the mobility of water, and reducing the size of ice crystals of beef patties during the repeated F-T cycles. In addition, FVP could effectively inhibited oxidation reaction and protein aggregation of beef patties with significant decreases in TBARS value, protein turbidity, contents of total sulfhydryl and carbonyl of myofibrillar protein, and an increase in protein solubility during the repeated cycles. These results suggest FVP could be developed to be a promising cryoprotectant in frozen patties.

Synthesis of trehalose-grafted poly(ester amide) with potential ice recrystallization inhibition activity

The ice recrystallization inhibition (IRI) activity of a macromolecular cryoprotectant is of great significance for applications in cryopreservation of biosamples. Herein, in order to create a possibly biodegradable trehalose-modified polymer, thiol-monosubstituted trehalose was synthesized by a simplified method, and then tethered to allylated-based poly(ester amide) (PEA) via thiol-ene click reaction. The synthesized PEA-g-trehalose (PEA-g-Tre) was able to inhibit ice recrystallization by 55% at 10 mg mL−1 compared to PBS control, superior to poly(ethylene glycol). Differential scanning calorimetry results showed that PEA-g-Tre exhibited a significantly higher glass transition temperature at 92.4 °C than PEA at 33.5 °C, and had a narrowed ice crystallization temperature range due to introduction of trehalose. Raman spectroscopy indicated that the IRI activity of PEA-g-Tre was attributed to the presence of trehalose residues in the side groups, which disrupted the regular arrangement of freezable water molecules. It is suggested that modification by trehalose can give rise to the IRI activity of the polymer through adsorption and diffusion mechanism with water, and thiol-ene reaction provides a feasible way for preparing polymeric cryoprotectants containing trehalose.

Antifreeze Polysaccharides from Wheat Bran: The Structural Characterization and Antifreeze Mechanism.

Exploring a novel natural cryoprotectant and understanding its antifreeze mechanism allows the rational design of future sustainable antifreeze analogues. In this study, various antifreeze polysaccharides were isolated from wheat bran, and the antifreeze activity was comparatively studied in relation to the molecular structure. The antifreeze mechanism was further revealed based on the interactions of polysaccharides and water molecules through dynamic simulation analysis. The antifreeze polysaccharides showed distinct ice recrystallization inhibition activity, and structural analysis suggested that the polysaccharides were arabinoxylan, featuring a xylan backbone with a majority of Araf and minor fractions of Manp, Galp, and Glcp involved in the side chain. The antifreeze arabinoxylan, characterized by lower molecular weight, less branching, and more flexible conformation, could weaken the hydrogen bonding of the surrounding water molecules more evidently, thus retarding the transformation of water molecules into the ordered ice structure.

Chitin nanocrystals – A new material with ice-shaping and ice recrystallization inhibition activities

Ice-binding materials that bind to ice and inhibit ice recrystallization can improve cell viability during cryopreservation and food quality during frozen storage. Recently several groups of amphiphilic materials were found to have ice recrystallization inhibition (IRI) activities. Like nanocelluloses, nanochitins are another group of natural materials demonstrating an amphiphilic characteristic. In this work, chitin nanocrystals (ChNCs) prepared by hydrochloride acid hydrolysis were studied for their ice-binding ability and ice recrystallization inhibition (IRI) activity. The ice-binding ability of ChNCs was demonstrated by an ice-shaping experiment at a concentration as low as 1 mg/g. In addition, the ChNCs were IRI active in sucrose and 1X phosphate-buffered saline (PBS) solutions at concentrations below 10 mg/g. Compared with the newly identified ice recrystallization inhibitor - cellulose nanocrystals (CNCs), ChNCs demonstrated much better ice-binding ability and IRI activity. Our data added ChNCs to the current list of ice-binding materials with IRI activity for many potential applications.

Optimizing alkylated ammonium-based compounds for ICE recrystallization inhibition (IRI) activity

Optimizing alkylated ammonium-based compounds for ICE recrystallization inhibition (IRI) activity

Self-Assembled Nanostructures of Homo-Oligopeptide as a Potent Ice Growth Inhibitor.

This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.

Succinylation of zein and gelatin hydrolysates improved their ice recrystallization inhibition activity.

The goal of this research was to enhance the ice recrystallization inhibition (IRI) activity of zein and gelatin hydrolysates (ZH and GH, respectively) by succinylation modification. ZH was prepared by Alcalase treatment for 3h and then modified by succinic anhydride (SA); whereas GH was made by Alcalase hydrolysis for 0.25h and succinylated by n-octylsuccinic anhydride (OSA). After 0.5h of annealing at -8°C at 40mg/mL, modified hydrolysates decreased the average Feret's diameter of ice crystal from 50.2μm (polyethylene glycol, negative control) to 28.8μm (SA modified ZH) and 29.5μm (OSA modified GH) in comparison to the unmodified hydrolysates, which had the crystal size of 47.2μm (ZH) and 45.4μm (GH). Also, the two succinylated samples had altered surface hydrophobicity, which potentially contributed to their enhanced IRI activity. Our results indicate that succinylation of food-derived protein hydrolysates can improve their IRI activity.

Hemp protein hydrolysates’ ability to inhibit ice recrystallization is influenced by the dispersing medium and succinylation

Freezing damage to foods can occur due to the formation of large ice crystals in the microstructure of foods, leading to unfavorable changes in the quality. The objective of this study was to determine the ice recrystallization inhibition (IRI) activity of hemp protein isolate (HPI) as affected by enzymatic hydrolysis and modification by succinylation. The IRI activity was evaluated using splat assay in phosphate buffer saline (PBS) and 10 mM NaCl. Enzymatic hydrolysis did not result in the production of IRI active hydrolysates when evaluated in PBS. Succinylation at 0.25:1 molar ratio of bromelain and trypsin hydrolysates resulted in the most IRI active hemp protein hydrolysates in PBS, having a 49% and 46% increase in IRI activity. When evaluated at 10 mM NaCl, both unmodified and modified HPI hydrolysates are IRI active. The unmodified hydrolysates were all significantly more IRI active in 10 mM NaCl, the hydrolysate produced by bromelain hydrolysis for 60 min had increased activity by 42% in the medium compared to PBS. Our results show that the ability of HPI hydrolysates to inhibit growth of ice crystal is influenced by the dispersing medium used during splat assay and succinylation.

Ice Recrystallization Inhibition Activity of Silk Proteins.

The cryopreservation of cells, tissue, and organs is essential in both fundamental research and practical applications, such as modern regenerative medicine and technological applications. However, the formation of ice crystals during ice recrystallization can have harmful or even fatal effects on biological systems. To address this challenge, we explore the ice recrystallization inhibition (IRI) activity of two natural silk proteins of Bombyx mori, fibroin and sericin. We found that silk fibroin (SF) had higher ice recrystallization inhibition activity than silk sericin (SS). Moreover, SF aqueous solutions perform better in inhibiting ice recrystallization than SF phosphate-buffered saline solutions. Sum-frequency generation spectroscopy shows that stronger electrostatic interactions are responsible for the higher IRI ability of SF. This work is significant for broadening the applications of silk proteins in biomedical fields.

Octenyl succinic anhydride-modified amyloid protein fibrils demonstrate enhanced ice recrystallization inhibition activity and dispersibility

Octenyl succinic anhydride (OSA) modification of amyloid proteins fibrils (APFs) was employed to improve dispersibility and ice recrystallization inhibition activity. OSA mainly reacted with the amino groups of APFs without significantly changing morphology. OSA-modified APFs (OAPFs) had lower pI, carried more negative charges, and were more hydrophobic. OSA-modification showed a pH-dependent effect on the dispersibility of fibrils. At pH 7.0, OSA-modification improved dispersibility and inhibited heat-induced gelation of fibrils at weakened electrostatic repulsion. OAPFs were more prone to aggregation with lower dispersity at acidic pH values and demonstrated stronger IRI activity than unmodified fibrils at pH 7.0. Our findings indicate OSA-modification favors the industrial application of APFs as an ice recrystallization inhibitor with enhanced dispersibility.