Ice Recrystallization Inhibition Activity Research Articles

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.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

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.

Poly(l-alanine-co-l-threonine succinate) as a Biomimetic Cryoprotectant.

We synthesized a series of [(l-Ala)x-co-(l-Thr succinate)y] (PATs), which are analogous to natural antifreezing glycoprotein with the structure of [l-Ala-l-Ala-l-Thr disaccharide]n, by varying the composition and degree of succinylation while fixing their molecular weight (Mn) and Ala/Thr ratio at approximately 10-12 kDa and 2:1, respectively. We investigated their ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), dynamic ice shaping (DIS), thermal hysteresis (TH), and protein cryopreservation activities. Both IRI and INI activities were greater for PATs with higher l-Ala content (PATs-3 and PATs-4) than those with lower l-Ala content (PATs-1 and PATs-2). DIS activity with faceted crystal growth was clearly observed in PATs-2 and PATs-4 with a high degree of succinylation. TH was small with <0.1 °C for all PATs and slightly greater for PATs with a high l-Ala content. Except for PATs-1, the protein (lactate dehydrogenase, LDH) stabilization activity was excellent for all PATs studied, maintaining LDH activity as high as that of fresh LDH even after 15 freeze-thaw cycles. To conclude, the cryo-active biomimetic PATs were synthesized by controlling the l-Ala content and degree of succinylation. Our results showed that PATs with an l-Ala content of 65-70% and degree of succinylation of 12-19% exhibited the cryo-activities of IRI, INI, and DIS, and particularly promising properties for the cryoprotection of LDH protein.

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.

Self-assembly of tamarind seed polysaccharide via enzymatic depolymerization and degalactosylation enhanced ice recrystallization inhibition activity

Polysaccharides are becoming potential candidates for developing food-grade cryoprotectants due to their extensive accessibility and health-promoting effects. However, unremarkable ice recrystallization inhibition (IRI) activity and high viscosity limit their practical applications in some systems. Our previous study found a galactoxyloglucan polysaccharide from tamarind seed (TSP) showing moderate IRI activity. Herein, the enhancement of the IRI performance of TSP via enzymatic depolymerization and degalactosylation-induced self-assembly was reported. TSP was depolymerized and subsequently removed ∼40 % Gal, which induced the formation of supramolecular rod-like fiber self-assembles and exhibited a severalfold enhancement of IRI. Ice shaping assay did not show obvious faceting of ice crystals, indicating that both depolymerized and self-assembled TSP showed very weak binding to ice. Molecular dynamics simulation confirmed the absence of molecular complementarity with ice. Further, it highlighted that degalactosylation did not cause significant changes in local hydration properties of TSP from the view of a single oligomer. The inconsistency between molecular simulation and macroscopic IRI effect proposed that the formation of unique supramolecular self-assemblies may be a key requirement for enhancing IRI activity. The findings of this study provided a new opportunity to enhance the applied potential of natural polysaccharides in food cryoprotection.

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.

Corn cob hemicelluloses as stabilizer for ice recrystallization inhibition in ice cream

Food stabilizers, such as guar gum and locust bean gum (LBG), are often added to ice cream to improve its texture and to combat its main shelf-life concern - ice recrystallization. Recently these gums have become increasingly expensive due to the limited supplies. In this study, holocellulose nanocrystals (holoCNCs) and hemicelluloses (hemiCs) were prepared from readily available corn cobs and tested for ice recrystallization inhibition (IRI) activities in the 25.0 % sucrose solution and ice cream mixes (ICMs). In the sucrose solution, holoCNCs were not IRI active at a concentration of 0.5 %, but hemiCs demonstrated a good IRI activity, even at 0.1 %. In the ICMs, the IRI activity of hemiCs was better than those of guar gum and LBG at a concentration of 0.2 %. Adding 0.2–0.5 % hemiCs had no negative influences on the physicochemical properties of ICMs and ice cream, including viscosity profile, particle size distribution, overrun, hardness, and meltdown rate. These research findings demonstrated corn cob hemiCs' potential as a more sustainable ice cream stabilizer.

Perturbation of Hydrogen Bonding Network Plays an Important Role in Determining Antifreeze Activity

In order to develop new antifreeze agents, it is crucial to understand the antifreeze activity. The adsorption-inhibition mechanism has been widely accepted, and most previous studies focus on ice-binding affinity of additive molecules but rarely discuss their influence on the hydrogen bonding network of ice. Herein, we investigate the ice recrystallization inhibition (IRI) activity of two structurally similar molecules, histidine (His) and phenylalanine (Phe). It is verified by experiment that Phe has excellent IRI activity, while His does not. Molecular dynamics simulation is conducted to study the process of ice growth with amino acids and to explore the origin of the difference between His and Phe. We find that His is prone to be swallowed up by ice and thus no longer hinders its growth, whereas Phe can remain unswallowed on the ice surface for a long period, which is attributed to their different abilities to perturb the hydrogen bonding network. Based on a statistical analysis of oxygen–hydrogen bond orientations, we propose a method to evaluate the order of the hydrogen bonding network. With this method, the IRI activity of His and Phe can be well interpreted, and the influence of different groups on the hydrogen bonding network can be quantified. This study takes the structural integrity of ice into consideration and provides a possibility to investigate the influence of different additive molecules on hydrogen bonding network, which is expected to improve the understanding of antifreeze mechanism.

Dimensional Reduction of Metal-Organic Frameworks for Enhanced Cryopreservation of Red Blood Cells.

To increase the red blood cell (RBC) cryopreservation efficiency by metal-organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra-thin metal-organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen-bonded water network and increased utilization of inner ordered structures, due to the higher surface-to-volume ratio. Specifically, a series of hafnium (Hf)-based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL-1 ) of thin-layered Hf-MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors.

Dimensional Reduction of Metal–Organic Frameworks for Enhanced Cryopreservation of Red Blood Cells

AbstractTo increase the red blood cell (RBC) cryopreservation efficiency by metal–organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra‐thin metal–organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen‐bonded water network and increased utilization of inner ordered structures, due to the higher surface‐to‐volume ratio. Specifically, a series of hafnium (Hf)‐based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL−1) of thin‐layered Hf‐MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors.