Ice-binding proteins: a remarkable ice crystal regulator for frozen foods.

Ice crystal growth during cold storage presents a quality problem in frozen foods. The development of appropriate technical conditions and ingredient formulations is an effective method for frozen food manufacturers to inhibit ice crystals generated during storage and distribution. Ice-binding proteins (IBPs) have great application potential as ice crystal growth inhibitors. The ability of IBPs to retard the growth of ice crystals suggests that IBPs can be used as a natural ice conditioner for a variety of frozen products. In this review, we first discussed the damage caused by ice crystals in frozen foods during freezing and frozen storage. Next, the methods and technologies for production, purification and evaluation of IBPs were summarized. Importantly, the present review focused on the characteristics, structural diversity and mechanisms of IBPs, and the application advances of IBPs in food industry. Finally, the challenges and future perspectives of IBPs are also discussed. This review may provide a better understanding of IBPs and their applications in frozen products, providing some valuable information for further research and application of IBPs.

Production, structure-function relationships, mechanisms, and applications of antifreeze peptides.

Growth of ice crystals can cause serious problems, such as frozen products deterioration, road damage, energy losses, and safety risks of human beings. Antifreeze peptides (AFPs), a healthy and effective cryoprotectant, have great potential as ice crystal growth inhibitors for a variety of frozen products. In this review, methods and technologies for the production, purification, evaluation, and characterization of AFPs are comprehensively summarized. First, this review describes the preparation of AFPs, including the methods of enzymatic hydrolysis, chemical synthesis, and microbial fermentation. Next, this review introduces the major methods by which to evaluate AFPs' antifreeze activity, including nanoliter osmometer, differential scanning calorimetry, splat-cooling, the biovaluation model, and novel technology. Moreover, this review presents an overview of the molecular characteristics, structure-function relationships, and action mechanisms of AFPs. Furthermore, advances in the application of AFPs to frozen food, including frozen dough, meat products, fruits, vegetable products, and dairy, are summarized and holistically analyzed. Finally, challenges of AFPs and future perspectives on their use are also discussed. An understanding of the production, structure-function relationships, mechanisms and applications of AFPs provides inspiration for further research into the use of AFPs in food science and food nutrition applications.

Cryopreservation of DNA Origami Nanostructures.

Although DNA origami nanostructures have found their way into numerous fields of fundamental and applied research, they often suffer from rather limited stability when subjected to environments that differ from the employed assembly conditions, that is, suspended in Mg2+ -containing buffer at moderate temperatures. Here, means for efficient cryopreservation of 2D and 3D DNA origami nanostructures and, in particular, the effect of repeated freezing and thawing cycles are investigated. It is found that, while the 2D DNA origami nanostructures maintain their structural integrity over at least 32 freeze-thaw cycles, ice crystal formation makes the DNA origami gradually more sensitive toward harsh sample treatment conditions. Whereas no freeze damage could be detected in 3D DNA origami nanostructures subjected to 32 freeze-thaw cycles, 1000 freeze-thaw cycles result in significant fragmentation. The cryoprotectants glycerol and trehalose are found to efficiently protect the DNA origami nanostructures against freeze damage at concentrations between 0.2 × 10-3 and 200 × 10-3 m and without any negative effects on DNA origami shape. This work thus provides a basis for the long-term storage of DNA origami nanostructures, which is an important prerequisite for various technological and medical applications.

Evaluation and preservation of vascular architectures in decellularized whole rat kidneys.

Organ transplantation is the gold standard treatment for end-stage organ failure. Due to the severe shortage of transplantable organs, only a tiny fraction of patients may receive timely organ transplantation every year. Decellularization-recellularization technology using allogeneic and xenogeneic organs is currently conceived to be a promising solution to generate functionally transplantable organs in vitro. This approach, however, still faces tremendous technological challenges, one of them being the ability to evaluate and preserve the integrity of vascular architectures upon decellularization and cryostorage of the whole organ matrices so that the off-the-shelf organ grafts are available on demand for clinical applications. In the present study, we report a Micro-CT imaging method for evaluating the integrity of vasculature of the decellularized whole organ scaffolds with/without freezing/thawing. The method uses radiopaque Microfil perfusion and x-ray fluoroscopy to acquire high-resolution angiography of the organ matrix. The whole rat kidney is decellularized using a new multistep perfusion protocol with the combined use of Triton X-100 and DNase. The decellularized kidney matrix is then cryopreserved after the pretreatment with different cryoprotectant solutions. The reconstructed tomographic images from Micro-CT confirm various structural alterations in the vasculature of the whole decellularized kidney matrix with/without frozen storage. The freezing damage to the vascular architectures can be reduced by perfusing cryoprotectant solutions into the whole kidney matrix. Ice-free cryopreservation with the vitrification solution VS83 can successfully preserve the integrity of the whole kidney matrix's vasculature after frozen storage.

Freeze-Damage Detection in Lemons Using Electrochemical Impedance Spectroscopy.

Lemon is the most sensitive citrus fruit to cold. Therefore, it is of capital importance to detect and avoid temperatures that could damage the fruit both when it is still in the tree and in its subsequent commercialization. In order to rapidly identify frost damage in this fruit, a system based on the electrochemical impedance spectroscopy technique (EIS) was used. This system consists of a signal generator device associated with a personal computer (PC) to control the system and a double-needle stainless steel electrode. Tests with a set of fruits both natural and subsequently frozen-thawed allowed us to differentiate the behavior of the impedance value depending on whether the sample had been previously frozen or not by means of a single principal components analysis (PCA) and a partial least squares discriminant analysis (PLS-DA). Artificial neural networks (ANNs) were used to generate a prediction model able to identify the damaged fruits just 24 hours after the cold phenomenon occurred, with sufficient robustness and reliability (CCR = 100%).

Assessing the potency and immunogenicity of inactivated poliovirus vaccine after exposure to freezing temperatures.

According to manufacturers, inactivated poliovirus vaccines (IPVs) are freeze sensitive and require storage between 2°C and 8°C, whereas oral poliovirus vaccine requires storage at -20 °C. Introducing IPV into ongoing immunization services might result in accidental exposure to freezing temperatures and potential loss of vaccine potency. To better understand the effect of freezing IPVs, samples of single-dose vaccine vials from Statens Serum Institut (VeroPol) and multi-dose vaccine vials from Sanofi Pasteur (IPOL) were exposed to freezing temperatures mimicking what a vaccine vial might encounter in the field. D-antigen content was measured to determine the in vitro potency by ELISA. Immunogenicity testing was conducted for a subset of exposed IPVs using the rat model. Freezing VeroPol had no detectable effect on in vitro potency (D-antigen content) in all exposures tested. Freezing of the IPOL vaccine for 7 days at -20 °C showed statistically significant decreases in D-antigen content by ELISA in poliovirus type 1 (p < 0.0001) and type 3 (p = 0.048). Reduction of poliovirus type 2 potency also approached significance (p = 0.062). The observed loss in D-antigen content did not affect immunogenicity in the rat model. Further work is required to determine the significance of the loss observed and the implications for vaccine handling policies and practices.

Early Detection of Freeze Damage in Navelate Oranges with Electrochemical Impedance Spectroscopy.

The early detection of freeze damage in Navelate oranges (Citrus sinensis L. Osbeck) was studied using electrochemical impedance spectroscopy (EIS), which is associated with a specific double-needle sensor. The objective was to identify this problem early in order to help to determine when a freeze phenomenon occurs. Thus, we selected a set of Navelate oranges without external defects, belonging to the same batch. Next, an intense cold process was simulated to analyze the oranges before and after freezing. The results of the spectroscopy analysis revealed different signals for oranges depending on whether they had experienced freezing or not. Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) of the obtained data demonstrated that it is possible to discriminate the samples, explaining 88.5% of the total variability (PCA) and being able to design a mathematical model with a prediction sensitivity of 80% (PLS-DA). Additionally, a designed artificial neural network (ANN) prediction model managed to correctly classify 100% of the studied samples. Therefore, EIS together with ANN-based data treatment is proposed as a viable alternative to the traditional techniques for the early detection of freeze damage in oranges.

Determination of freeze damage on HPV vaccines by use of flow cytometry.

The human papillomavirus (HPV) vaccines Gardasil, Silgard and Cervarix were labeled with antibodies against HPV strain 6 or 16/FITC conjugated secondary antibodies and analyzed by flow cytometry. The vaccines showed distinct peaks of fluorescent particles, and a shift towards decreased fluorescent particles was observed after incubation of the vaccines over night at -20 °C. Since parallel distributed vaccines could have longer route of transportation there is an increased risk of freeze damage for these types of vaccine. Shift in fluorescence of labeled vaccine particles was used to indicate whether parallel distributed Silgard, which is a vaccine type identical to Gardasil, was exposed to freeze damage during transportation, but no shift was observed. Additional experiments showed that the HPV vaccines could be degraded to smaller particles by citric acid/phosphate buffer treatment. The majority of particles detected in degraded Gardasil were very small indicating that the particles are HPV virus like particle (VLPs) labeled with antibodies, but Cervarix could only be degraded partially due to the presence of another type adjuvant in this vaccine. The described method may be useful in characterization of adjuvanted vaccines with respect to freeze damage, and to characterize vaccines containing particles corresponding to VLPs in size.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.

Sedimentation behavior of quality and freeze-damaged aluminum-adjuvanted vaccines by wNMR.

Vaccine sedimentation and resuspension are properties that vaccine makers use to characterize a suspension product during research and development as well as throughout the shelf life of the vaccine. Three vaccines with three different aluminum adjuvants and different antigens were selected and monitored over the course of sedimentation using water proton nuclear magnetic resonance (wNMR) relaxometry. This simple method measured fully intact, single-dose vaccine vials and reported sedimentation profiles for each, which readily distinguished freeze-stressed vaccines from unstressed vaccines.

Optical cryomicroscopy and differential scanning calorimetry of buffer solutions containing cryoprotectants.

In the pharmaceutical industry, cryoprotectants are added to buffer formulations to protect the active pharmaceutical ingredient from freeze- and thaw damage. We investigated the freezing and thawing of aqueous sodium citrate buffer with various cryoprotectants, specifically amino acids (cysteine, histidine, arginine, proline and lysine), disaccharides (trehalose and sucrose), polyhydric alcohols (glycerol and mannitol) and surfactants (polysorbate 20 and polysorbate 80). Hereby, we employed optical cryomicroscopy in combination with differential scanning calorimetry in the temperature range to -80 °C. The effect of cryoprotectants on the morphology of the ice crystals, the glass transition temperature and the initial melting temperature is presented. Some of the cryoprotectants have a significant impact on ice crystal size. Disaccharides restrict ice crystal growth, whereas surfactants and glycerol allow ice crystals to increase in size. Cysteine and mannitol cause dehydration after thawing. Either one or two glass transition temperatures were detected, where arginine, surfactants, glycerol, proline and lysine suppress the second, implying a uniform freeze-concentrated solution. The initial melting temperature of pure buffer solution can be shifted up by adding mannitol, both disaccharides and both surfactants; but down by glycerol, proline and lysine.

Assessment of freeze damage in fruits and vegetables.

Freezing is an efficient and widely used method of food preservation. However, it can also cause irreversible damages at cellular level which in turn degrade the overall quality of the frozen food products. Therefore, qualitative and quantitative methods and technologies that will be able to evaluate with accuracy the freeze damage are of great importance. This review paper provides a comprehensive study of the methods that have been used to evaluate the freeze damage in fruits and vegetables. Further than the principles and the applications of those methods, the advantages and the limitations are also being discussed.