Use Of Antifreeze Proteins Research Articles

Antifreeze Proteins: Novel Applications and Navigation towards Their Clinical Application in Cryobanking.

Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future.

Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades

Antifreeze proteins (AFPs) are synthesized by diverse non-mammalian species, allowing them to survive in severely cold environments. Since the 1990s, the scientific literature reports their use for low-temperature preservation of germplasm. The aim of this systematic review was to compile available scientific evidence regarding the use of AFP for low-temperature preservation of several reproductive specimens. Internet databases were consulted using the terms: “antifreeze protein” OR “AFP” OR “antifreeze glycoprotein” OR “AFGP” OR “ice-binding protein” OR “IBP” OR “thermal hysteresis protein” AND “cryopreservation”. From 56 articles, 87 experiments testing AFPs in low-temperature preservation of gametes, embryos or reproductive tissues/cells were fully analyzed and outcomes were annotated. A positive outcome was considered as a statistically significant improvement on any parameter evaluated after low-temperature preservation with AFP, whereas a negative outcome included worsening of any evaluated parameter, in comparison to untreated groups or groups treated with a lower concentration of AFP. The findings indicated that research on the use of AFP as a cryoprotectant for reproductive specimens has increased markedly over the past decade. Some experiments reported both positive and negative results, which depended, on AFP concentration in the preservation media. Variation in the outcomes associated with species was also observed. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes after the use of AFP. In fishes, positive and negative outcomes were observed in 71.4% and 33.3% of 21 experiments, respectively. Most positive outcomes included preserving cell post-warming survival. The beneficial effect of AFP supports its use in cryobiological approaches used in human and veterinary medicines and animal protein industry. Moreover, combination of different AFP types, or AFP with antioxidants, or even the use of AFP-biosimilar, comprise some promising approaches to be further explored in cryopreservation.

Addition of antifreeze protein type I or III to extenders for ram sperm cryopreservation

Antifreeze proteins (AFP) play an important role in cellular survival at sub-zero temperatures. This study assessed the effect of AFP type I or III in semen extender (TRIS-egg yolk) for ram sperm cryopreservation. Pooled semen of four rams were allocated into five treatments: Control (CONT, without AFP); AFP Type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) μg/mL]; or III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) μg/mL], and then frozen in six replicates. Treatments affected kinetic parameters, plasma membrane integrity and morphology (P < 0.05). The AFPIII-0.1 presented lesser total motility. Linearity was greater in AFPI-0.1, AFPI-0.5 and AFPIII-0.5 and straightness was greater in all AFP-supplemented extenders. Plasma membrane integrity was greater in AFPI-0.1 and AFPI-0.5. All AFP groups had greater percentage of normal sperm than CONT. No differences (P > 0.05) were observed in hypoosmotic test, sperm acrosome status, mitochondrial activity, chromatin condensation, perivitelline membrane binding rate and lipoperoxidation. In conclusion, the use of AFP, predominantly type I, may increase sperm cell protection during cryopreservation, with no adverse effect on potential fertilization capacity or increase in reactive oxygen species, being a potential cryoprotectant to ram sperm.

The use of antifreeze proteins to modify pore structure in directionally frozen alginate sponges for cartilage tissue engineering

It is thought that osteoarthritis is one of the world’s leading causes of disability, with over 8.75 million people in the UK alone seeking medical treatment in 2013. Although a number of treatments are currently in use, a new wave of tissue engineered structures are being investigated as potential solutions for early intervention. One of the key challenges seen in cartilage tissue engineering is producing constructs that can support the formation of articular cartilage, rather than mechanically inferior fibrocartilage. Some research has suggested that mimicking structural properties of the natural cartilage can be used to enhance this response. Herein directional freezing was used to fabricate scaffolds with directionally aligned pores mimicking the mid-region of cartilage, anti-freeze proteins were used to modify the porous structure, which in turn effected the mechanical properties. Pore areas at the tops of the scaffolds were 180.46 ± 44.17 μm2 and 65.66 ± 36.20 μm2 for the AFP free and the AFP scaffolds respectively, and for the bases of the scaffolds were 91.22 ± 19.05 μm2 and 69.41 ± 21.94 μm2 respectively. Scaffolds were seeded with primary bovine chondrocytes, with viability maintained over the course of the study, and regulation of key genes was observed.

The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos.

The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present in several organisms exposed to subzero temperatures. The ability for these proteins to inhibit ice recrystallization together with their ability to interact with biological membranes makes them interesting molecules to be used in cryopreservation protocols. This mini-review provides a general overview about the use of antifreeze proteins to improve the short and long term storage of gametes and embryos.

Application of Antifreeze Proteins in Foods of Animal Origin- A Review

This paper reviews the origin, functionality and use of antifreeze proteins (AFPs) in frozen foods of animal origin. AFPs inhibit recrystallization and reduce cellular damage, thus safeguarding the quality of frozen foods without structural and organoleptic alterations. The conventional food cryoprotectants are of synthetic/chemical origin and may be hazardous to health. In spite of the limitations like cost and time involvement for small scale purification, the AFPs of natural origin are attracting the interest of researchers for their mass commercial production and application in frozen food. A small amount of purified AFPs are sufficient to exhibit activity without much taste alterations, owing to its commercial benefit. Therefore, naturally occurring AFPs can be used as potent cryopreservatives in frozen meat, fish and dairy products to minimize the structural and functional damages during freezing and thawing.

Antifreeze Proteins: Characteristics, Function, Mechanism of Action, Sources and Application to Foods

Antifreeze proteins (AFPs) are synthesized by various organisms to enable their cells to survive subzero environments. AFPs exert their effects by lowering the freezing point of water as well as preventing the growth of ice crystals and recrystallization during frozen storage. Their ability to modify the growth of ice results in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice, which could reduce drip loss during thawing, improve the quality and extend the shelf life of frozen food. However, the high cost of these proteins and the requirement of biotechnological methods for commercial production limit their potential use in foods. Despite these limitations relatively small amounts are sufficient to exhibit activity, which suggests AFPs as possible additives for foods in the future. This review presents the results of investigations on the characteristics, function, mechanism of action, sources and potential food uses of AFPs. Practical Applications Antifreeze proteins (AFPs) act both by lowering the freezing point of water and by inhibiting ice crystals, and prevent recrystallization during storage. Because of their advantageous characteristics in recent years, numerous studies have been made on the use of AFPs in improving the properties of frozen foods. The aim of this paper is to present the results of numerous investigations on AFPs obtained from fish, plants or insects and discuss their characteristics, function, mechanism of action, sources and potential food uses.

Revisão: Proteínas anticongelantes – uma tecnologia emergente para o congelamento de alimentos

Um dos métodos mais tradicionais na conservação de alimentos, o congelamento também pode alterar de forma significativa as características do produto. Grandes cristais de gelo provocam alteração na textura e/ou danos a membranas e componentes celulares. As técnicas de congelamento rápido formam cristais de gelo menores do que o processo lento, porém as flutuações de temperatura durante a distribuição e transporte podem promover o crescimento dos cristais. Esse processo é conhecido como recristalização e é uma barreira na utilização do congelamento como método de conservação em muitos casos. O uso de crioprotetores tradicionais, como a sacarose, é uma alternativa limitada, uma vez que concentrações elevadas são requeridas. Na década de 1970, foi descrita em peixes de águas frias uma classe de proteínas que, em baixa concentração, pode interagir e influenciar o crescimento do cristal de gelo. Elas foram chamadas de proteínas anticongelantes (PACs), sendo encontradas também em plantas, animais e micro-organismos ambientados a baixas temperaturas. Essas proteínas podem intervir no processo de formação do núcleo inicial do gelo, reduzir o ponto de congelamento da água, ou, ainda, inibir a recristalização, principalmente para PACs de vegetais. Há diversos trabalhos publicados e algumas patentes registradas para o uso de PACs em diversos alimentos, como lácteos, carnes, massas, frutas e hortaliças, conservando de melhor forma as características originais do alimento. Atualmente, o custo ainda é uma barreira para utilização comercial das PACs. Contudo, a descoberta de novas fontes pode reduzir seu custo e tornar essas proteínas uma ferramenta efetiva na manutenção da textura de alimentos congelados. Baseada em trabalhos que avaliaram aspectos químicos das PACs e exemplos de sua aplicação, esta revisão tem como objetivo principal apresentar as características gerais das PACs e discutir sobre sua utilização.

The use of antifreeze proteins for frost protection in sensitive crop plants

Antifreeze proteins (AFPs), also known as ice binding proteins (IBPs), have evolved as an important adaptation in numerous organisms exposed to subzero temperatures. Plant AFPs have only been identified in freeze tolerant species (those able to survive extracellular freezing). Consequently, plant AFPs have very low specific activities as they have not evolved to completely prevent ice formation in the plant. In contrast, fish and most insect AFPs function to prevent freezing in species that have evolved freeze avoidance mechanisms. Therefore, the activity of these AFPs, especially those of insects (as they are generally exposed to considerably lower temperatures than fish), is much greater. The ability of AFPs to non-colligatively lower the freezing point of water (thermal hysteresis) has led to the idea that frost-sensitive crop plants could avoid damage resulting from common minor frost events in late spring and early autumn by expressing high activity AFPs that permit them to remain unfrozen to temperatures of approximately −5°C. Over the past 20 years, the efficacy of this concept has been tested in a variety of studies that produced transgenic plants (including Arabidopsis thaliana, and several crop plants) expressing various AFPs. Initially, fish AFPs were employed in these studies but as insect AFPs, with higher levels of antifreeze activity, were discovered these have become the AFPs of choice in plant transformation studies. Some studies have produced transgenic plants that have exhibited improved cold tolerance of 1–3°C compared to the wild-type. None of the studies with transgenic plants, however, have yet attained a sufficient level of protection. Progress to this point indicates that more significant results are achievable. If so, the billions of dollars lost annually to frost damage of sensitive crops could be avoided. Geographic ranges and growing seasons could also be expanded. This review provides an overview of the studies of transgenic plants producing AFPs, and makes suggestions for future advancements in this field of study.

141 Inhibition of ice recrystallization by antifreeze proteins

Ice recrystallization (IR) is a process in which large ice crystals grow at the expense of smaller ones. It occurs constantly in nature and in industrial processes in conditions of moderate cooling of a partially frozen environment or at accelerated rate due to temperature fluctuations of frozen substances. IR is one of the major causes of smooth texture loss and deterioration of frozen food such as ice cream during storage. In cryopreservation, recrystallization during thawing, damages the cells and tissues due to membrane rapture and cell dehydration, reducing the survival rates. Organisms inhabiting cold environments and prone to IR injuries, such as freeze-tolerating plants and freeze-avoiding organisms like fish and insects have developed antifreeze proteins (AFPs), a subset of the Ice Binding Proteins group which interact with ice for various biological purposes. The AFPs adsorb to ice surfaces and restrict the growth of ice to the areas between bound protein molecules. Such proteins were shown to block IR effectively [Knight and Duman, 1986], and may serve as additives in many applications in which ice recrystallization is an obstacle. Although ice recrystallization inhibition (IRI) activity of a variety of AFPs was studied [Knight et al., 1995], the mechanism by which AFPs inhibit IR is unknown. Particularly, it has been shown that although insect AFPs have considerably higher freezing point depression activity relative to AFPs from fish and plants, their IRI activity is in the same range or lower [Yu et al., 2010]. We intend to study the mechanism of IRI in order to explain the inconsistency between this activity and the freezing point depression activity of insect AFPs. In our study the IRI activity of AFPs from mealworm Tenebriomolitor (TmAFP) was investigated. Sucrose solutions containing various concentration of TmAFP were cooled to −50 °C, then the temperature was elevated and sustained constant for annealing. During annealing, images of recrystallization were acquired using a light microscope equipped with fluorescence illumination and cold-stage. Initial results show an accumulation of TmAFP onto ice crystals and IRI in a range of AFPs concentrations. Our next goals are to develop test methods that will provide reliable, quantitative information on recrystallization and on the accumulation of the protein on the crystals, as well as depletion of the protein from the solution. Such methods will enable quantification of the ice recrystallization phenomena and subsequently quantification of the differences between the IRI activities of various types of AFPs. Understanding the mechanism of ice recrystallization and the effects of AFPs on it may improve and expand the use of AFPs in many applications in the medical sector, in cryopreservation, and in the frozen food industry. Source of funding: European Research Council (ERC). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

Food Processing Technology in a Sustainable Food Supply Chain

The development of sustainable process technologies was a major concern and a central idea of what Prof. W.E.L. Spiess (WELS) and his research group have tried to accomplish throughout a long and productive scientific journey. In the early sixties, Freeze Drying was considered as the process with the lowest thermal impact on quality of food. WELS recognized the importance of both freezing process and storage conditions (especially water activity) for maintaining a high product quality. His group laid the foundation for a better understanding of water sorption in food materials. In fact, their critical evaluation of the BET theory initiated fruitful discussions on water sorption in foods. Their work on freezing included studies on crystal growth conditions, the impact of Time-Temperature conditions in the Frozen Food Chain (FFC), the development of various Time-Temperature Indicators and the use of antifreeze proteins in frozen food production. They actually developed the “mother” of all electronic Time-Temperature Recorders; that was an electronic temperature data-logger used for the analysis of product temperatures and residence times in the FFC. The techniques they developed in their studies of the crystallization process allowed the WELS group to develop an osmotic dehydration cell attached to a Confocal Scanning Laser Microscope (CSLM). This arrangement allowed them to understand and accurately describe the mass transfer process at cellular level during osmotic dehydration. Throughout his productive scientific career Prof. Spiess coordinated several European research cooperation projects that were instrumental in the development of a European Network of Food Science/Engineering Institutions and a true “European Food Science community”.

Advances In Food Freezing/Thawing/Freeze Concentration Modelling and Techniques

This paper reviews recent developments on various aspects of food freezing. The various ef fects of freezing and frozen storage on food quality are described, including the role of glass transition. Methods for calculating freezing time and the evolution of temperature and phase change are reviewed and their underlying assumptions and limitations are critically examined. Finally, recent developments in freezing, thawing and freeze concentration techniques are reviewed: high pressure freezing and thawing, ultrasound assisted freezing, progressive freeze concentration, osmodehydrofreezing, immersion freezing in ice slurry, and the use of antifreeze proteins.

Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 μg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1–10 μg/ml compared to the control ( P < 0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 μg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.

Anin VivoStudy of Antifreeze Protein Adjuvant Cryosurgery

Cryosurgery employs freezing to destroy undesirable tissue. However, under certain thermal conditions, frozen tissues survive. The survival of frozen undesirable tissue may lead to complications, such as recurrence of cancer. In a study of nude mice with subcutaneous metastatic prostate tumors, we showed that the preoperative injection of a phosphate-buffered saline solution with 10 mg/ml antifreeze protein of type I into the tumor prior to freezing enhances destruction under thermal conditions which normally yield cell survival. This suggests that the adjunctive use of antifreeze proteins in cryosurgery may reduce the complications from undesirable tissues that survive freezing.