Antifreeze Activity Research Articles

Unveiling the mechanism of high-molecular-weight glutenin subunit deletions at the Glu-B1 locus affecting gluten deterioration during dough frozen storage and freeze-thaw cycles: An integrative experimental and in silico study

This study explored how high-molecular-weight glutenin subunits (HMW-GS) at the Glu-B1 locus impact gluten deterioration during dough frozen storage and freeze-thaw (F/T) cycles. Using deletion lines, we found that the deletion of specific HMW-GS, particularly Bx7, led to a greater reduction in glutenin macropolymer (GMP) wet weight during storage, especially under F/T cycles. The frozen conditions triggered more dissociation of hydrogen and disulfide bonds, generating more protein monomers and resulting in severe gluten deterioration in the Bx7 deletion. Additionally, protein structures in these lines were more vulnerable to damage during F/T cycles due to temperature fluctuations. In silico analysis further confirmed that Bx7 had better stability and antifreeze activity compared to By8, explaining why its deletion had a more pronounced effect on gluten stability. These findings offer significant implications for the food industry, particularly in enhancing the quality, shelf-life, and commercial viability of frozen dough products by providing a deeper understanding of the mechanisms behind gluten deterioration, specifically the role of high-molecular-weight glutenin subunits.

Solvation of molecules from the family of "domain of unknown function" 3494 and their ability to bind to ice.

In 2012, the molecular structure of a new, broad class of ice-binding proteins, classified as "domain of unknown function" (DUF) 3494, was described for the first time. These proteins have a common tertiary structure and are characterized by a very wide spectrum of antifreeze activity (from weakly active to hyperactive). The ice-binding surface (IBS) region of these molecules differs significantly in its structure from the IBS of previously known antifreeze proteins (AFPs), showing a complete lack of regularity and high hydrophilicity. The presence of a regular, repeating structural motif in the IBS region of hitherto known AFP molecules, combined with the hydrophobic nature of this surface, promotes the formation of an ice-like ordering of the solvation water layer and, as a result, facilitates the process of transformation of this water layer into ice. It is, therefore, surprising that the newly discovered DUF3494 class of proteins clearly breaks out of this characteristic. In this paper, using molecular dynamics simulations, we analyze the solvation water structure of the IBS region of both DUF3494 family molecules and AFPs. As we show, although the IBS of DUF3494 molecules does not form an ice-like water structure in the solvation layer, this is compensated by the formation of the equivalent of "anchored clathrate water," in the form of a relatively large number of water molecules bound to the surface of the protein molecule and providing potential binding sites for it to the ice surface.

Expeditious chemical synthesis of xylomannans disproves the proposed antifreeze activities.

Cold-adapted species are able to generate cryoprotective proteins and glycoproteins to prevent freezing damage. The [→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→] n xylomannan from the Alaska beetle Upis ceramboides was disclosed by Walters and co-workers in 2009 as the first glycan-based antifreeze agent, which was later reported to be found in diverse taxa. Here, we report the rapid synthesis of four types of xylomannans, including the proposed antifreeze xylomannan up to a 64-mer (Type I), the regioisomeric [→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→] n 16-mer (Type II), the diastereomeric [→4)-β-L-Manp-(1→4)-β-D-Xylp-(1→] n 16-mer (Type III) and the block-wise [→4)-β-D-Manp-(1→] m [→4)-β-D-Xylp-(1→] n 32-mer (Type IV), by employing a strategic iterative exponential glycan growth (IEGG) process. The nuclear magnetic resonance spectral data of the alleged natural xylomannan are in accordance only to those of the block-wise Type IV glycan and none of these synthetic xylomannans has been found to be capable of inducing thermal hysteresis. These results disprove the previous reports about the natural occurrence of antifreeze xylomannans.

Effect of ultrasonic pretreatment on antifreeze activity of gluten antifreeze polypeptide

In this study, the effects of thermal hysteresis activity, ice recrystallization inhibition, amino acid composition and surface hydrophobicity of different ultrasonic pretreatments on gluten antifreeze polypeptide was studied.The results of the thermal hysteresis activity showed that the collaborative (flat-panel combined probe) ultrasonic working mode was superior to the single-frequence ultrasound mode. In the optimal working mode (15 min, 100 W/L), the results of polarized light microscope and gel chromatography column showed that ultrasonic pretreatment could effectively inhibit the growth of ice crystals, reduced the size of ice crystals, and to a certain extent, transfer the molecular weight distribution of peptides to small molecular peptides. The molecular weight was mainly concentrated at about 200–1000 u. The contents of Valine, Methionine, Leucine, Proline and Glutamic acid residues in the gluten antifreeze polypeptide pretreated by ultrasonic wave were relatively high and more hydrophobic groups were exposed. The secondary structure results showed that the molecular conformation of the polypeptide was mainly α-helix and β-sheet conformations.This paper systematically revealed the intrinsic correlation mechanism between the changes of key domains of substrate proteins and the antifreeze activity of enzymatic hydrolysis products assisted by ultrasoud, providing a theoretical basis for the preparation of antifreeze polypeptide.

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.

Identification and isolation of a novel antifreeze peptide from crayfish shells

In this study, a novel antifreeze peptide was isolated from crayfish shells and its antifreeze mechanism and activity were investigated. Firstly, the crayfish shells peptides (CSPs) were prepared by Enzymatic Hydrolysis Technology (alkaline protease and trypsin) and the CSPs could significantly enhance the survival rate of Saccharomyces cerevisiae from 12.5% to 88.5% after 24h freeze-thaw cycle. Then, a total 1004 peptides were identified from CSPs by liquid chromatography/tandem mass spectrometry (LC-MS/MS), among those peptides, two peptide sequences YWDHPIRDGFAPH (AFP1) and GPPGKPGIPDIVDW (AFP1) were selected as potential antifreeze polypeptides (AFPs) according the physicochemical properties: instability index, hydrophilicity, and confidence level. Furthermore, the antifreeze activity and mechanism of AFP1 and AFP2 were studied through molecular dynamics simulation, revealing that the AFP1 and AFP2 could adsorb to ice surface with 7 and 14 hydrogen bonds, respectively. Moreover, AFP2 has a more stable binding capacity and could interact with 33 water molecules. At last, the results of differential scanning calorimetry (DSC) and cold-stage polarization microscope indicated that the thermal hysteresis (TH) values of AFP2 were 1.62–2.09 °C and could inhibit ice recrystallization. Therefore, this study provides a theoretical basis for developing safe peptide-based cryoprotectants which will improve the quality of frozen foods.

Improvement of the antifreeze activity of tilapia skin collagen peptides through enzymatic supramolecular assembly approach

SummaryEnzymatic supramolecular assembly was applied to increase the thermal hysteresis activity (THA) of tilapia skin collagen peptides and their applicability as cryoprotectants. The THA value of the glutamine‐assembled product increased by 61.5% and its ice crystal content dropped by 33.93% compared to the collagen peptides. Following initial separation and purification, the THA of Gln‐AFPs was further elevated by 28.6%. After five freeze–thaw cycles, tilapia surimi treated with 8% Gln‐AFPs had high levels of salt‐soluble protein, total sulfhydryl content, Ca2+‐ATPase activity, and water‐holding capacity. Additionally, the cryoprotective effect of Gln‐AFPs on the surimi was found to be superior to that of commercial antifreeze. The majority of the Gln‐AFPs turns folded, according to FTIR analysis, and ‐helices were produced. After supramolecular assembly, the Gln‐AFPs structure also smoothed out. As a result, the enzymatic supramolecular assembly drastically increased the antifreeze activity of the tilapia skin‐collagen peptides, which could eventually be developed into a novel antifreeze additive in the food industry.

Synthetic Antifreeze Glycoproteins with Potent Ice-Binding Activity.

Antifreeze glycoproteins (AFGPs) are produced by extremophiles to defend against tissue damage in freezing climates. Cumbersome isolation from polar fish has limited probing AFGP molecular mechanisms of action and limited development of bioinspired cryoprotectants for application in agriculture, foods, coatings, and biomedicine. Here, we present a rapid, scalable, and tunable route to synthetic AFGPs (sAFGPs) using N-carboxyanhydride polymerization. Our materials are the first mimics to harness the molecular size, chemical motifs, and long-range conformation of native AFGPs. We found that ice-binding activity increases with chain length, Ala is a key residue, and the native protein sequence is not required. The glycan structure had only minor effects, and all glycans examined displayed antifreeze activity. The sAFGPs are biodegradable, nontoxic, internalized into endocytosing cells, and bystanders in cryopreservation of human red blood cells. Overall, our sAFGPs functioned as surrogates for bona fide AFGPs, solving a long-standing challenge in accessing natural antifreeze materials.

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.

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.

Recent advances, challenges and functional applications of antifreeze protein in food industry

SummaryFreezing is a common food preservation method that can extend the shelf life of food, but the ice crystals and recrystallisation during freezing, storage and transportation cause significant damage to frozen food. Antifreeze proteins are a class of proteins that widely exist in organisms living in cold conditions, such as fish, insects, plants and microorganisms in Antarctica and other cold regions. Antifreeze proteins can reduce the freezing point of water, inhibit recrystallisation effects, modify ice crystal morphology and effectively suppress the quality deterioration caused by ice crystals during food freezing. This review comprehensively introduces the source, antifreeze mechanism, application in food, influencing factors and regulating methods of antifreeze activity of antifreeze proteins, as well as the limitations of antifreeze proteins. It also looks forward to the future application of antifreeze proteins in food.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

The antifreeze activity and physicochemical properties ofLitopenaeus vannameihead autolysate

SummaryLitopenaeus vannameiheads were autolysed at a constant temperature of 50 °C, pH 7.0 for a maximum duration of 5 h, and the antifreeze activity and physicochemical properties of the head autolysates were determined. Thermal hysteresis (TH) was used as an index for determining the antifreeze activity of the shrimp head autolysates. The highest thermal hysteresis activity was 1.82 °C which was measured in the 5 h‐shrimp head autolysate. The highest negative zeta potential value (−41.06 ± 2.08 mV) and surface hydrophobicity (295.575 ± 9.7819) were in the 5 and 1.5 h autolysate groups, respectively. Generally, &lt;2000 Da components accounted for over 85% of the total molecular weight in all shrimp head autolysate groups. Pearson correlation analysis was used to investigate how physicochemical properties influenced the thermal hysteresis index. Although at varying degrees, the analysis confirmed that a positive correlation existed between TH activity and molecular weight, hydrophobic amino acid content, and surface hydrophobicity. A negative correlation existed between TH activity and zeta potential, and hydrophilic amino acids. The findings of our study suggest thatLitopenaeus vannameihead autolysate has a potential antifreeze effect and that the physicochemical properties influence its thermal hysteresis.

Mechanism of antifreeze protein functioning and the "anchored clathrate water" concept.

In liquid water, there is a natural tendency to form aggregates that consist of water molecules linked by hydrogen bonds. Such spontaneously formed aggregates are surrounded by a "sea" of disordered water molecules, with both forms remaining in equilibrium. The process of creating water aggregates also takes place in the solvation water of proteins, but in this case, the interactions of water molecules with the protein surface shift the equilibrium of the process. In this paper, we analyze the structural properties of the solvation water in antifreeze proteins (AFPs). The results of molecular dynamics analysis with the use of various parameters related to the structure of solvation water on the protein surface are presented. We found that in the vicinity of the active region responsible for the binding of AFPs to ice, the equilibrium is clearly shifted toward the formation of "ice-like aggregates," and the solvation water has a more ordered ice-like structure. We have demonstrated that a reduction in the tendency to create "ice-like aggregates" results in a significant reduction in the antifreeze activity of the protein. We conclude that shifting the equilibrium in favor of the formation of "ice-like aggregates" in the solvation water in the active region is a prerequisite for the biological functionality of AFPs, at least for AFPs having a well-defined ice binding area. In addition, our results fully confirm the validity of the "anchored clathrate water" concept, formulated by Garnham et al. [Proc. Natl. Acad. Sci. U. S. A. 108, 7363 (2011)].

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17°C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

Co-functional Group Conducting Hydrogels Inspired by Ligament for Flexible Electronic Devices.

The application of conductive hydrogels in flexible electronics has attracted much interest in recent years due to their excellent mechanical properties and conductivity. However, the development of conductive hydrogels combining with superior self-adhesion, mechanical properties, antifreeze, and antibacterial activity is still a challenge. Herein, inspired by the structure of the ligament, a multifunctional conductive hydrogel is constructed to address the issue by introducing collagen into the polyacrylamide. The obtained conductive hydrogel exhibits outstanding conductivity (52.08 mS/cm), ultra-stretchability (>2000%), self-adhesion, and antibacterial properties. More significantly, the supercapacitor based on this hydrogel electrolyte achieves a desirable capacitance (514.7 mF·cm-2 at 0.25 mA·cm-2 current density). As a wearable strain sensor, the obtained hydrogel can rapidly detect different movements of the body such as finger, wrist, elbow, and knee joints. It is conceived that this study would provide a potential approach for the preparation of conductive hydrogels in the application of flexible electronics.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Rational Design of and Mechanism Insight into an Efficient Antifreeze Peptide for Cryopreservation

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge. Here, we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins (IBPs) with evolution analysis. In this way, several peptides with notable antifreeze activity were developed. In particular, a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition (IRI), solubility, and biocompatibility, making it suitable for use as a cryoprotective agent (CPA). A mutation analysis and molecular dynamics (MD) simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity, while the Ser18 residue can synergistically enhance their interaction with ice, revealing the antifreeze mechanism of AVD. Furthermore, to evaluate the cryoprotection potential of AVD, the peptide was successfully employed for the cryopreservation of various cells, which demonstrated significant post-freezing cell recovery. This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

An Antibacterial andAnti-Oxidative Hydrogel Dressing for Promoting Diabetic Wound Healing and Real-Time Monitoring Wound pH Conditions with a NIR Fluorescent Imaging System.

The design and synthesis of multifunctional chitosan hydrogels based on polymerized ionic liquid and a near-infrared (NIR) fluorescent probe (PIL-CS) is a promising strategy, which not only prevents the transition from acute to chronic wounds, but also provides prompt measures regarding microenvironmental alterations in chronic wounds. PIL-CS hydrogel can real-time visualize wound pH through in vivo NIR fluorescent imaging and also feature the pH-responsive sustained drug release, such as antioxidant, to eliminate reactive oxygen species (ROS) and to boost diabetic wound healing. PIL-CS hydrogel is specific, sensitive, stable, and reversible in response to pH changes at the wound site. It, therefore, enables real-time monitoring for a dynamic pH change in the microenvironment of irregular wounds. PIL-CS hydrogel is also designed to possess many merits including high water containment and swelling rate, good biocompatibility, electrical conductivity, antifreeze, tissue adhesion, hemostatic performance, and efficient antibacterial activity against MRSA. In vivo studies showed that PIL-CS hydrogel provided fast diabetic wound healing support, promoted vascular endothelial growth factor (VEGF) production, and reduced ROS and tumor necrosis factor (TNF-α) generation. The results support that the hydrogels coupled with NIR fluorescent probes can be an excellent diabetic wound dressing for enhancing and real-time monitoring skin restoration and regeneration.

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.