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.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

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.     

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.     

Isolation of viable cell mass from frozen <Emphasis Type="Italic">Microcystis viridis</Emphasis> bloom containing microcystin-RR

Cyanobacterial species commonly occur in the phytoplankton of freshwater lakes and sometimes develop as toxin-producing blooms. Microcystis is one of the most common genera of freshwater cyanobacteria and is often the dominating phytoplankton of eutrophic lakes all over the world. In eutrophic lakes, large amounts of Microcystis may overwinter in the sediment and re-inoculate the water column in spring. In most cases, the overwintering pelagic population—if it exists—is small, and its role in re-inoculation has not been clear yet. In December 2005, we found large amounts of Microcystis on the surface, frozen in the ice cover in a eutrophic pond (Pond Hármashegy, Hungary). We identified the Microcystis species and investigated the viability and the toxicity of the frozen cells. The dominant species in the bloom samples was Microcystis viridis. Viability tests showed that the colonies isolated from the ice cover were composed of living cells. The isolated strain was found toxic, we analyzed the microcystin composition in the frozen planktonic Microcystis mass; in the investigated samples microcystin-RR was the main cyanotoxin.     

The effect of wintering sites on the survival and reproduction of Gyraulus acronicus (Gastropoda) in a partly frozen river

Summary In a boreal river about 95% of the individuals of Gyraulus acronicus overwinter in the littoral zone which freezes solid each year. These snails were compared with those overwintering in the unfrozen sublittoral area: The littoral snails had a higher survival rate, a higher tissue dry mass/CaCO₃ ratio, and they deposited a higher number of eggs. Littoral snails had a more pronounced tissue degrowth during winter. High winter survival in the frozen littoral zone, a refuge totaly free from predation, indicates that overwintering here is advantageous. However, during frozen periods of short duration (<1 month) the high initial mortality to which the snails were exposed when freezing into the ice was not compensated for by higher survival after the initial phase. Under such conditions when the frozen period is very short the snails would have higher survival in unfrozen parts and are thus expected to avoid the ice.     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Responses of Microbial Communities in Arctic Sea Ice After Contamination by Crude Petroleum Oil

Microbial communities associated with Arctic fjord ice polluted with petroleum oils were investigated in this study. A winter field experiment was conducted in the Van Mijen Fjord (Svalbard) from February to June 2004, in which the ice was contaminated with a North Sea paraffinic oil. Holes were drilled in the ice and oil samples frozen into the ice at the start of the experiment. Samples, including cores of both oil-contaminated and clean ice, were collected from the field site 33, 74, and 112 days after oil application. The sampled cores were separated into three sections and processed for microbiological and chemical analyses. In the oil-contaminated cores, enumerations of total prokaryotic cells by fluorescence microscopy and colony-forming units (CFU) counts of heterotrophic prokaryotes both showed stimulation of microbial growth, while concentrations of oil-degrading prokaryotes remained at similar levels in contaminated and clean ice. Analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) revealed that bacterial communities in oil-contaminated ice generated fewer bands than communities in clean ice, although banding patterns changed both in contaminated and clean ice during the experimental period. Microbial communities in unpolluted ice and in cores contaminated with the paraffinic oil were examined by cloning and sequence analysis. In the contaminated cores, the communities became predominated by Gammaproteobacteria related to the genera Colwellia, Marinomonas, and Glaciecola, while clean ice included more heterogeneous populations. Chemical analysis of the oil-contaminated ice cores with determinations of n-C17/Pristane and naphthalene/phenanthrene ratios indicated slow oil biodegradation in the ice, primarily in the deeper parts of the ice with low hydrocarbon concentrations.     

Evaluation of extenders and cryopreservatives for cooling and cryopreservation of spermatozoa from the western spotted skunk (Spilogale gracilis)

Experiments were conducted to develop a suitable protocol for cryopreservation of spotted skunk semen. Semen was collected by electroejaculation of captive male skunks (n = 16) from late January through late November. In the first experiment, fresh semen was diluted in either TEST (n = 10), TRIS (n = 9), or BF5F (n = 7) extenders and maintained at 4°C for 16 hr. Sperm motility in these extenders was not significantly different before cooling (P = 0.71), but samples diluted with BF5F exhibited significantly lower sperm motility than the other extenders at all time points after cooling (P < 0.05). In the second experiment, fresh semen was diluted in TEST containing either 3, 5, or 10% DMSO or 3, 5, or 10% glycerol as a cryopreservative. These samples were cooled to 4°C and frozen in 0.25 ml French straws on dry ice. Some samples containing 5% DMSO or 5% glycerol (n = 4), were also frozen on dry ice as pellets. Frozen samples were maintained in liquid nitrogen. Fresh samples had significantly greater sperm motility in dimethyl sulfoxide (DMSO) than in glycerol (P < 0.05), while frozen and thawed samples had the highest motility in 5 or 10% DMSO or 10% glycerol. Samples frozen in French straws had significantly greater sperm motility after freezing and thawing than those frozen by the pellet method (P < 0.05). Optimum cryoprotection was achieved with the TEST extender containing 5 or 10% DMSO, when used in conjunction with French straws. © 1992 Wiley-Liss, Inc.     

Intra- and inter-cores fungal diversity suggests interconnection of different habitats in an Antarctic frozen lake (Boulder Clay,Northern Victoria Land)

A perennially frozen lake at Boulder Clay site (Victoria Land, Antarctica), characterized by the presence of frost mounds, have been selected as an in situ model for ecological studies. Different samples of permafrost, glacier ice and brines have been studied as a unique habitat system. An additional sample of brines (collected in another frozen lake close to the previous one) was also considered. Alpha- and beta-diversity of fungal communities showed both intra- and inter-cores significant (p < 0.05) differences, which suggest the presence of interconnection among the habitats. Therefore, the layers of frost mound and the deep glacier could be interconnected while the brines could probably be considered as an open habitat system not interconnected with each other. Moreover, the absence of similarity between the lake ice and the underlying permafrost suggested that the lake is perennially frozen based. The predominance of positive significant (p < 0.05) co-occurrences among some fungal taxa allowed to postulate the existence of an ecological equilibrium in the habitats systems. The positive significant (p < 0.05) correlation between salt concentration, total organic carbon and pH, and some fungal taxa suggests that a few abiotic parameters could drive fungal diversity inside these ecological niches.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Soy sauce increased the oxidative stress tolerance of nematode via p38 MAPK pathway

Soy sauce – a fermented food made from soybeans and wheat – is considered a healthy seasoning, but little scientific evidence is available to support this. In this study, physiological effects of soy sauce were analyzed using Caenorhabditis elegans. When soy sauce was fed to C. elegans together with Escherichia coli OP50, fat accumulation decreased, and resistance to oxidative stress by H₂O₂ was greatly increased in the nematodes. qRT-PCR revealed that mRNA expression of oxidative stress tolerance genes, including sod, ctl, and gpx, was markedly increased in soy sauce-fed nematodes. Worms ingesting soy sauce showed high mitochondrial membrane potential and reactive oxygen species (ROS) and low intracellular ROS, suggesting that soy sauce induced mitohormesis and decreased cytoplasmic ROS. Therefore, soy sauce ingestion affects the mitochondria and may alter the fat metabolism in C. elegans. Furthermore, the increase in oxidative stress tolerance is mediated through p38 MAPK pathway.     

Expeditious prediction of post-mortem changes in frozen fish meat using three-dimensional fluorescence fingerprints

The present study was conducted to characterize fluorophores in the fish body using three-dimensional fluorescence fingerprints (3D-FFs) and to utilize these 3D-FFs obtained from frozen horse mackerel (Trachurus japonicus) fillets to predict early post-mortem changes. Alive fish were sacrificed instantly, preserved in ice until 2 days, and then filleted, vacuum packed, and frozen. Subsequently, 3D-FFs of the frozen fillets were acquired using F-7000 aided with a fiber probe. Post-mortem freshness changes were tracked by measuring adenylate energy charge (AEC) values and nicotinamide adenine dinucleotide (NAD and NADH) content. Partial least squares regression models for predicting AEC values and NADH content in frozen fish meat showed good fittings, with R² of 0.90 and 0.85, by utilizing eight and five excitation wavelengths, respectively, based on their fluorescence features acquired from standard fluorophores. This novel approach of 3D-FFs could be utilized as an efficient technique for at-line monitoring of frozen fish quality.     

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion‐weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high‐temperature exotherm (?3°C to ?5°C) corresponded with ice formation and high ice‐nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (?6°C and ?8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low‐temperature exotherm corresponded with SAM injury and the killing temperature in wheat (?21°C) and rye (?27°C). The SAM had lower ice‐nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.     

Physiological adaptations to low temperature and brine exposure in the circumpolar amphipod Gammarus wilkitzkii

Summary The amphipod Gammarus wilkitzkii does not survive being frozen totally into solid sea ice. When the animals are cooled in air or freezing seawater, they will freeze and die at a temperature of about-4° C. However, during sea ice growth, the amphipods may tolerate to stay in the vicinity of the ice by conforming to the ambient brine in a salinity range of 34 ppt to about 60 ppt. A passive relationship between the concentrations of the haemolymph and seawater Na⁺ and Cl^-, lowers the melting point of the body fluids of the animals, thus preventing internal ice formation at low temperatures.     

Evidence for Ideal and Non-Ideal Equilibrium Freezing of Leaf Water in Frosthardy Ivy (Hedera helix) and Winter Barley (Hordeum vulgare)

Temperature dependences of leaf water potentials (ψ_leaf) of frozen leaves of frosthardy ivy and winter barley were determined psychrometrically and found to coincide with the respective water potentials of ice which were obtained using the same technique. The water potentials of ice showed good agreement with theoretically established data. Analysis of the components of ψ of frozen leaves of Hedera helix revealed ideal equilibrium freezing, i.e. the governing of the relative content of liquid (or frozen) water solely by the osmotic potential. In winter barley, by contrast, a negative pressure potential was demonstrated to contribute to ψ_leaf. even under conditions of moderate frost. This reduced the degree of protoplast dehydration and the extent to which the concentrations of the cellular solutes rose. Such a freezing behavior is termed non-ideal equilibrium freezing. Depending on the original content of leaf water, the volume increments of liquid water due to the negative pressure potential amounted up to 10% at ?6 °C and even more at a lower temperature. In addition to the experimental data, a theoretical treatment of psychrometry at subzero temperatures is presented.     

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.     

Effect of pretreatment with paste and sauce extract made using Tenebrio molitor larvae on ethanol‐damaged HepG2 cells

In this study, we made koji using protein‐rich Tenebrio molitor larvae (TML) inoculated with Aspergillus oryzae, and then used the koji to prepare a paste and a sauce. The TML koji showed the highest amino nitrogen, protease activity, and free amino acids content when it was fermented for 72 h after inoculation with 0.8% A. oryzae. The koji was aged in 20% saltwater for 50 days, and then the paste and sauce were separated. To evaluate the effect of TML paste and sauce on ethanol (EtOH)‐damaged hepatocytes, aspartate aminotransferase (AST) and alanine aminotransaminase (ALT) were measured in EtOH‐treated HepG2 cells after pretreatment with TML paste and sauce extract. As a result, we confirmed that TML paste and sauce extracts lowered the AST and ALT content in the medium, compared to soybean sauce and paste extracts. TML paste and sauce extract significantly reduced the expression of tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, a biomarker of inflammation, and significantly increased the activity of the antioxidants superoxide dismutase (SOD) and reduced glutathione (GSH) in proportion to the amount of TML added to the paste and sauce. These results suggest that the intake of TML paste and sauce, a new type of fermented food made from insects, may provide effective protection to the liver against hepatocyte injury by EtOH via anti‐inflammatory and antioxidative effects.     

Thermal, Mechanical, and Molecular Relaxation Properties of Frozen Sucrose and Fructose Solutions Containing Hydrocolloids

Model frozen systems formulated with 20wt% sucrose or fructose and with the addition of 0.3 or 0.5wt% of xanthan gum (XG), guar gum (GG), locust bean gum (LBG), or a 50wt% mixture of XG and LBG were studied by differential scanning calorimetry, dynamic mechanical analysis, and ¹H-pulsed nuclear magnetic resonance. Melting onset of either the sucrose or fructose model systems was not affected by the addition of hydrocolloids. As expected, ice content was lower in fructose than in sucrose systems. Addition of hydrocolloids had no effect on ice content, except when the blend of XG and LBG was added to the fructose system, where ice content was significantly diminished. Hydrocolloids decreased molecular mobility for both frozen sucrose or fructose solutions, especially for the addition of XG/LBG blend. Relaxation times and storage modulus of the frozen systems with added hydrocolloids were significantly lower than the control frozen sugar solutions.