Calorimetric and Microstructural Investigation of Frozen Hydrated Gluten

The thermal and microstructural properties of frozen hydrated gluten were studied by using differential scanning calorimetry (DSC), modulated DSC, and low-temperature scanning electron microscopy (cryo-SEM). This work was undertaken to investigate the thermal transitions observed in frozen hydrated gluten and relate them to its microstructure. The minor peak that is observed just before the major endotherm (melting of bulk ice) was assigned to the melting of ice that is confined to capillaries formed by gluten. The Defay–Prigogine theory for the depression of melting point of fluids confined in capillaries was put forward in order to explain the calorimetric results. The pore radius size of the capillaries was calculated by using four different empirical models. Kinetic analysis of the growth of the pore radius size revealed that it follows first-order kinetics. Cryo-SEM observations revealed that gluten forms a continuous homogeneous and not fibrous network. Results of the present investigation showed that is impossible to assign a T _g value for hydrated frozen gluten because of the wide temperature range over which the gluten matrix vitrifies, and therefore the construction of state diagrams is not feasible at subzero temperatures for this material. Furthermore, the gluten matrix is deteriorated with two different mechanisms from ice recrystallization, one that results from the growth of ice that is confined in capillaries and the other from the growth of bulk ice.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Antifreeze proteins and their potential use in frozen foods

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each organism. As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many foods consumed as part of the human diet. However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer.     

Relationship between Recrystallization Rate of Ice Crystals in Sugar Solutions and Water Mobility in Freeze-Concentrated Matrix

To better understand the relation between recrystallization rate and water mobility in freeze-concentrated matrix, isothermal ice recrystallization rates in several sugar aqueous solutions and self-diffusion coefficients of water component in corresponding freeze-concentrated matrix were measured. The sugars used were fructose, glucose, maltose, and sucrose. The sugar concentrations and temperature were varied so that ice contents for all samples were almost equal. Neither recrystallization rates nor diffusion coefficients depended uniformly on temperature. The recrystallization rates increased with increasing the diffusion coefficients, and a direct relationship was found between recrystallization rate and diffusion coefficient. This indicated that self-diffusion coefficient of water component in freeze-concentrated matrix is a useful parameter for predicting and controlling recrystallization rate in sugar solutions relevant to frozen desserts.     

Estimation of Water Diffusion Coefficients in Freeze-Concentrated Matrices of Sugar Solutions Using Molecular Dynamics: Correlation Between Estimated Diffusion Coefficients and Measured Ice-Crystal Recrystallization Rates

The diffusion coefficient of the water component in a freeze-concentrated matrix is a useful parameter for predicting and controlling the recrystallization rate of ice crystals in sugar solutions relevant to frozen desserts. Herein, application of molecular dynamics (MD) for estimating the water diffusion coefficient in a freeze-concentrated matrix of sugar solutions is described. Diffusion coefficients evaluated using MD with the optimized potentials for liquid simulations all atom force field and water models of three types (simple point charge, simple point charge extended, and transferable intermolecular potential-4 point) show a good positive linear relation with measured values, indicating that the MD methods used in this study are useful for predicting differences in water diffusion coefficients in a sugar freeze-concentrated matrix. Furthermore, similarly to measured values, the estimated diffusion coefficients show a good positive correlation with recrystallization rates of ice crystals, which suggests that MD is useful to predict differences in recrystallization rates of ice crystals in frozen sugar solutions.     

Quantitative cryomicroscopic analysis of intracellular freezing of granulocytes without cryoadditive

Purified human granulocytes were frozen in isotonic saline at different constant cooling rates down to -60 degrees C and subsequently thawed on the thermally defined cryostage of a cryomicroscope. Cells monitored on videotape were examined with respect to cooling rate threshold, type, and temperature of intracellular ice formation during cooling and recrystallization during warming. Two apparently different mechanisms of intracellular ice formation (iif) were distinguished during cooling, i.e., "twitching" (no visible ice front) and "darkening" (diffuse ice front). Both types of iif are related to cooling rate and hence also to dehydration. Cooling rate thresholds and temperatures of intracellular recrystallization were determined. It was found that twitching iif occurs just about 6.3 to 7.4 degrees C above the homogeneous nucleation temperature, suggesting that it might be catalyzed by nucleators present within the cells. Darkening iif, on the other hand, was observed at much higher temperatures, i.e., 23.4 to 28.3 degrees C above the homogeneous nucleation temperature, which could possibly indicate a nucleation induced by extracellular ice crystals (at a cooling rate of 30 degrees K/min, however, darkening iif was observed to occur at a temperature lower than that required for twitching iif). The proposed mechanisms of cryoinjury are related to membrane integrity measurements presented in M. W. Scheiwe, Ch. K?rber, and S. Englich, Cryo-Letters, 5, 300-306, 1984.     

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

Effects of warming rate,temperature, and antifreeze proteins on the survival of mouse spermatozoa frozen at an optimal rate

We have recently reported that the survival of mouse spermatozoa is decreased when they are warmed at a suboptimal rate after being frozen at an optimal rate. We proposed that this drop in survival is caused by physical damage derived from the recrystallization of extracellular ice during slow warming. The first purpose of the present study was to determine the temperatures over which the decline in survival occurs during slow warming and the kinetics of the decline at fixed subzero temperatures. The second purpose was to examine the effects of antifreeze proteins (AFP) on the survival of slowly warmed mouse spermatozoa, the rationale being that AFP have the property of inhibiting ice recrystallization. With respect to the first point, a substantial loss in motility occurred when slow warming was continued to higher than -50 degrees C and the survival of the sperm decreased with an increase in the temperature at which slow warming was terminated. In contrast, the motility of sperm that were warmed rapidly to these temperatures remained high initially but dropped with increased holding time. At -30 degrees C, most of the drop occurred in 5 min. These results are consistent with the hypothesis that damage develops as a consequence of the recrystallization of the external ice. AFP ought to inhibit such recrystallization, but we found that the addition of AFP-I, AFP-III, and an antifreeze glycoprotein at concentrations of 1-100 microg/ml did not protect the frozen-thawed cells; rather it led to a decrease in survival that was proportional to the concentration. There was no decrease in survival from exposure to the AFP in the absence of freezing. AFP are known to produce changes in the structure and habit of ice crystals, and some have reported deleterious consequences associated with those structural changes. We suggest that such changes may be the basis of the adverse effects of AFP on the survival of the sperm, especially since mouse sperm are exquisitely sensitive to a variety of mechanical stresses.     

Understanding the Influence of State/Phase Transitions on Ice Recrystallization in Atlantic Salmon (Salmo salar) During Frozen Storage

Temperature fluctuations during storage and distribution of frozen foods lead to ice recrystallization and microstructural modifications that can affect food quality. Low temperature transitions may occur in frozen foods due to temperature fluctuations, resulting in less viscous and partially melted food matrices. This study systematically investigated the influence of state/phase transitions and temperature fluctuations on ice recrystallization during the frozen storage of salmon fillets. Using a modulated differential scanning calorimeter, we identified the characteristics glass transition temperature (T _g ′) of −27 °C and the onset temperature for ice crystal melting (T _m ′) of −17 °C in salmon. The temperature of salmon fillets in sealed plastic trays was lowered to −35 °C in a freezer to achieve the glassy state. The temperature (T) of frozen salmon fillets in sealed plastic trays was modulated to achieve a rubbery state (T > T _m ′), a partially freeze-concentrated state (T _g ′ < T < T _m ′) and a glassy state (T < T _g ′). We performed temperature modulation experiments by exposing packaged salmon to room temperature twice a day for 2 to 26 min during 4 weeks of storage. We also analyzed ice crystal morphology using environmental scanning electron microscopy and X-ray computed tomography techniques to observe the pore distribution after sublimation of ice crystals. Melt–refreeze and isomass rounding mechanisms of ice recrystallization were noticed in the frozen salmon subjected to temperature modulations. Results show that ice crystal growth occurred even in the glassy state of frozen salmon during storage, with or without temperature fluctuations. Ice crystal size in frozen salmon was greater in the rubbery state (T > T _m ′) due to the increased mobility of unfrozen water compared to the glassy state. The morphological/geometric parameters of ice crystals in frozen salmon stored for 1 month differed significantly from those in 0-day storage. These findings are important to the frozen food industry because they can help optimize storage and distribution conditions and minimize quality loss of frozen salmon due to recrystallization.     

An ice-binding protein from an Antarctic sea ice bacterium

An Antarctic sea ice bacterium of the Gram-negative genus Colwellia, strain SLW05, produces an extracellular substance that changes the morphology of growing ice. The active substance was identified as a approximately 25-kDa protein that was purified through its affinity for ice. The full gene sequence was determined and was found to encode a 253-amino acid protein with a calculated molecular mass of 26,350 Da. The predicted amino acid sequence is similar to predicted sequences of ice-binding proteins recently found in two species of sea ice diatoms and a species of snow mold. A recombinant ice-binding protein showed ice-binding activity and ice recrystallization inhibition activity. The protein is much smaller than bacterial ice-nucleating proteins and antifreeze proteins that have been previously described. The function of the protein is unknown but it may act as an ice recrystallization inhibitor to protect membranes in the frozen state.     

Ice recrystallization in a model system and in frozen muscle tissue

Recrystallization produces modifications on ice crystal sizes during storage and transport of frozen foods, reducing the advantages obtained by quick freezing and inducing physicochemical changes which alter their quality and shorten their shelf life. This process involves the growth of the larger crystals at the expense of the smaller ones, being the interfacial energy, the driving force of the phenomenon. In the present work recrystallization was analyzed using direct microscopic observation of ice crystals in a model solution (0.28 N NaCl) and indirect observation of frozen muscle tissue. The model solution allowed visualization of the interface behavior; from the analysis of the ice crystal frequency distributions, relationships between shape and size of the grains were established. A kinetic model based on the average system curvature was proposed obtaining a satisfactory fitness of the experimental data. Values of the kinetic constants determined at different temperatures allowed the estimation of the process activation energy. In muscle tissues isothermal freeze-substitution was used to observe the holes left by the ice in frozen semitendinous beef muscle stored at -5, -10, -15, and -20 degrees C during long periods of time. A different evolution of the mean ice crystal diameter was observed with respect to the model system. In meat samples, at long storage times, a limit diameter value was reached; this situation has been proved to be independent of temperature and initial size (freezing rate); a theoretical expression based on tissue characteristic parameters was proposed for its evaluation. Activation energy for recrystallization in muscle tissue was also determined, being comparable to values for protein denaturation and quality losses.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.

     

Overwintering of benthic macroinvertebrates in ice and frozen sediment in a North Swedish river

In winter the water freezes into the substrate within considerable areas of unregulated northern rivers due to low temperature combined with a lowering of the water level. Living individuals of Nematoda, Gastropoda, Sphaeriidae, Oligochaeta, Hirudinea, Isopoda, Trichoptera and Chironomidae were found in samples of ice and frozen sediment from the bottom frozen hydrolittoral zone of the north Swedish river Vindelälven. All abundant species in the frozen substratum, except Asellus aquaticus , seemed to be well adapted to withstand overwintering in this special habitat free from predation. Generally, between 80 and l00% of enclosed animals survived thawing. Cysts or other kinds of resting stage constructions, similar to those found during drought, were common in several enclosed species. Specimens of Gyraulus acronicus, Pisidium ssp., Molanna albicans and Chironomidae survived exposure to −4°C for five month in a freezing experiment. Extracellular freezing of the invertebrates overwintering in the ice is probable, as the ambient temperature was below the true freezing point of most animals. The composition of the substratum may effect the survival of animals enclosed in ice.     

Bioprospecting for microbial products that affect ice crystal formation and growth

At low temperatures, some organisms produce proteins that affect ice nucleation, ice crystal structure, and/or the process of recrystallization. Based on their ice-interacting properties, these proteins provide an advantage to species that commonly experience the phase change from water to ice or rarely experience temperatures above the melting point. Substances that bind, inhibit or enhance, and control the size, shape, and growth of ice crystals could offer new possibilities for a number of agricultural, biomedical, and industrial applications. Since their discovery more than 40 years ago, ice nucleating and structuring proteins have been used in cryopreservation, frozen food preparation, transgenic crops, and even weather modification. Ice-interacting proteins have demonstrated commercial value in industrial applications; however, the full biotechnological potential of these products has yet to be fully realized. The Earth’s cold biosphere contains an almost endless diversity of microorganisms to bioprospect for microbial compounds with novel ice-interacting properties. Microorganisms are the most appropriate biochemical factories to cost effectively produce ice nucleating and structuring proteins on large commercial scales.     

Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.     

Intracellular ice formation and growth in MCF-7 cancer cells

Direct cell injury in cryosurgery is highly related to intracellular ice formation (IIF) during tissue freezing and thawing. Mechanistic understanding of IIF in tumor cells is critical to the development of tumor cryo-ablation protocol. In aid of a high speed CMOS camera system, the events of IIF in MCF-7 cells have been studied using cryomicroscopy. Images of ‘darkening’ type IIF and recrystallization are compared between cells frozen with and without ice seeding. It is found that ice seeding has significant impact on the occurrence and growth of intracellular ice. Without ice seeding, IIF is observed to occur over a very small range of temperature (∼1 °C). The crystal dendrites are indistinguishable, which is independent of the cooling rate. Ice crystal grows much faster and covers the whole intracellular space in comparison to that with ice seeding, which ice stops growing near the cellular nucleus. Recrystallization is observed at the temperature from −13 °C to −9 °C during thawing. On the contrary, IIF occurs from −7 °C to −20 °C with ice seeding at a high subzero temperature (i.e., −2.5 °C). The morphology of intracellular ice frozen is greatly affected by the cooling rate, and no ‘darkening’ type ice formed inside cells during thawing. In addition, the intracellular ice formation is directional, which starts from the plasma membrane and grows toward the cellular nucleus with or without ice seeding. These results can be used to explain some findings of tumor cryosurgery in vivo, especially the causes of insufficient killing of tumor cells in the peripheral area near vessels.     

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220?mM solutions of disaccharides; however, the best cell viability was obtained when a 200?mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.     

Ice Recrystallization Behavior of Corn Starch/Sucrose Solutions: Effects of Addition of Corn Starch and Antifreeze Protein III

Knowledge of the behavior of corn starch during frozen storage is necessary to understand more complex systems. In the present study, ice recrystallization in corn starch (0.3% and 3%, w/w)/sucrose (40%, w/w) solution was investigated at −10 °C based on the theory of Ostwald ripening. The addition of corn starch to the sucrose solution increased the ice recrystallization (IR) rate constant. To explore the mechanism causing higher IR rate constant, fluorescence microscopy was used to analyze the distribution of corn starch molecules. Fluorescence micrograph showed corn starch distributed homogenously in the freeze-concentrated phase. Ice crystal size distribution assessment showed that at the same average radius, the addition of corn starch increased the standard deviation of ice crystal size distribution. The findings revealed that the addition of corn starch widened the distribution of ice crystal size, which may be the mechanism causing higher IR rate constant. To inhibit the ice recrystallization process, antifreeze protein type III (AFP III) was added to sucrose solutions with and without corn starch. In the presence of corn starch, 0.01-mg/mL AFP III was enough to significantly reduce the IR rate. Conversely, the samples without corn starch did not show a significant reduction in IR rate constant at the same AFP III concentration. The outcomes revealed that corn starch enhanced the activity of AFP III. The results of this study showed that corn starch increased the IR rate constant, and AFP III supplemented with corn starch was synergistically more efficient in retarding IR rate constant.

     

The mechanism of cryohemolysis: by direct observation with the cryomicroscope and the electron microscope.

Blood films (3–8 μm thick) supported between two glass coverslips were frozen to ?20 °C. In the extracellular areas, ice cavities of the order of 0.2 μm separated by bands of dense plasma were evident when examined with the electron microscope; intracellular ice was not observed with the light microscope. Electron microscopy also showed the presence of intracellular ice particles of the order of 0.2–0.7 μm, these appeared as fine reticulations when observed with the light microscope. Upon gradual rewarming the following changes were observed: recrystallization in the extracellular matrix (?18 to ?8 °C), intracellular recrystallization (?13 to ?10 °C), transfer of water from erythrocytes to extracellular areas (?9 to ?7 °C), and melting and hemolysis (?6 to ?2 °C).Freezing of blood at ?3 °C and subsequent thawing did not cause hemolysis of the red cells. In blood frozen at ?3 °C and cooled to ?20 °C or frozen by abrupt exposure to 20 °C the erythrocytes hemolyzed in 7/16–11/16 of a second, whereas in blood frozen at ?3 °C and cooled to ?10 °C the cells hemolyzed in 5–15 sec even though the mode if lysis (i.e., uniform seepage of hemoglobin from the surface of the cell) was similar in all cases. This indicates that the presence of intracellular ice does not seem to play a major role in the injury to the erythrocytes. The mechanism of cryoinjury demonstrated by hemolysis has been discussed.