The “Intrinsic” Thermal Conductivity of Some Wet Proteins in Relation to Their Hydrophobicity: Analysis on Gelatin Gel

Since the intrinsic thermal conductivity of protein cannot be measured directly, the apparent “intrinsic” thermal conductivity of gelatin was estimated on the basis of the most suitable heat conduction model. Among four models of heat conduction through heterogeneous material, the series layers model best described the experimental results for frozen gelatin gels of different concentrations. Then, the “intrinsic” thermal conductivity of frozen wet gelatin was estimated to be 0.61 [W/m·°C]. The “intrinsic” thermal conductivity of unfrozen wet gelatin was estimated on the basis of Filippov’s equation to be 0.38 [W/m·°C], since the unfrozen gelating gel seemed to be as homogeneous as the solution.     

Sydonol,a New Fungal Morphogenic Substance Produced by an Unidentified Aspergillus sp.

The “intrinsic” thermal conductivity values of unfrozen wet egg-albumin, wheat gluten and milk casein were determined on the basis of the series heat conduction model to be 0.238, 0.219 and 0.200 [W/m·°C], respectively. The corresponding values for frozen samples were 0.403, 0.315 and 0.273 [W/m·°C], respectively. The “intrinsic” thermal conductivity values of wet proteins determined in the previous and present studies were between the thermal conductivity values of water (or ice) and oils (or fats), in the reverse order of the average hydrophobicity values of proteins.     

Measurement of the Thermal Conductivity of Unfrozen and Frozen Food Materials by a Steady State Method with Coaxial Dual-cylinder Apparatus

Coaxial dual-cylinder apparatus was used to measure the effective thermal conductivity of aqueous solutions of glucose, sucrose, gelatin and egg albumin over a temperature range from –20° to 20°C by the steady state method. The accuracy of the apparatus was confirmed by testing with water and ice. The effective thermal conductivity decreased with an increase in the total solid content in both the frozen and unfrozen states. In the unfrozen state, the effective thermal conductivity was slightly dependent on temperature. In the frozen state, however, the effective thermal conductivity was strongly dependent on temperature; lower temperatures gave higher effective thermal conductivity, reflecting the increase in the ice fration. For the unfrozen samples, the intrinsic thermal conductivity of each solid component was calculated by heat transfer models. All the models tested, series, parallel and Maxwell–Eucken, were equally applicable to describe the heat conduction in the unfrozen state. In the frozen state, however, the strong temperature dependency of the effective thermal conductivity suggests that the effect of the temperature dependency of the ice fraction should be incorporated into theoretical models.     

Deterioration of Antarctic Krill Muscle during Freeze Storage

The effects of freezing on the heat-induced gelation, Ca²⁺-ATPase activity and myofibril structure of Antarctic krill muscle were investigated. Muscle from freshly caught krill was immediately stored at-20°C in the presence (to prevent freezing) (glycerol krill) and absence (frozen krill) of glycerol. Several protease inhibitors, monosodium glutamate and Ca²⁺ were individually added to glycerol krill to inhibit endogenous proteolysis. The examinations described above were carried out after about 3-month storage at-20°C. In glycerol krill (unfrozen state), viscoelastic parameters of the heat-induced gels and Ca²⁺-ATPase activities of all the krill samples were similar to those of “surimi” (raw fish meat paste) of Alaska pollack which gave gel of good quality, although the micro structure (Z-lines) of myofibrils was different among the glycerol krill samples. In frozen krill, however, the parameters of the gel were different from those of “surimi”, the ATPase activity was completely lost and disruption of the myofibril structure occurred. Refreezing (-20°C) of glycerol krill after removal of glycerol resulted in a marked decrease in the gelation ability. These results suggest that freezing of krill muscle causes deterioration of the gelation ability.     

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of [³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.     

Laser Raman investigation of intact single muscle fibers on the state of water in muscle tissue

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nulilus). The spectra in the region of the O-H (or O-²H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of “structured” intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 ± 0.01 for H₂O and 1.05 ± 0.03 for ²H₂O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state at ?5°C, which corresponds to 1 g of water per of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.     

Protein-bound SH groups in frozen-thawed egg cells of the sea urchin

Unfertilized egg cells of the sea urchin St. intermedius could survive slow freezing to ?15 °C for a short period of time, but at the same freezing temperature extracellular freezing became fatal within a few hours. Such freezing injury resulted in “black” or “white” cytolysis in frozen-thawed cells. “Black” cytolysis took place in the process of both freezing and thawing, while “white” cytolysis occurred only on thawing. Rapid rewarming consistently produced “white” cytolysis in extracellularly frozen cells. The observed behavior of the injured cells during freeze-thawing appeared favorable for the explanation of freezing injury by the SH-SS hypothesis. Protein-bound SH groups were quantitatively determined in both whole cell and cortex with plasma membrane before and after freeze-thawing. However, no significant change in the SH value was observed between freeze-thaw cytolysed materials and unfrozen ones.     

A Newer Method for Isolation of the 7S Globulin in Soybean Seeds

The effects of freezing on the proteolysis of beef during storage at 4°C after being thawed was investigated.

A sarcoplasmic 32-kDa protein in frozen as well as unfrozen beef decreased rapidly during storage at 4°C, and a more than 100-kDa protein appeared in both beef samples. And the increment of peptides in the frozen beef during the storage at 4°C was larger than that in the unfrozen beef, suggesting that the proteolysis was faster during the storage of the former than the latter. However, its increment in the frozen beef for 10 weeks during the storage at 4°C became smaller than that of the one frozen for less than 5 weeks.

To discover an indicator for evaluation of the conditioning of frozen and unfrozen beef, peptides produced during the storage of beef at 4°C were surveyed. A peptide, APPPPAEVPEVHEEV, was detected and seemed to be available as an indicator in the conditioning of beef.     

Microwave thawing of frozen kidneys: A theoretically based experimentally-effective design

Equipment was designed and fabricated for uniformly thawing frozen canine kidneys using single-frequency electromagnetic radiation. Complete and uniform warming of frozen kidneys from ?70 to +14 °C over periods ranging from 1.5 to 4.5 min was achieved without “cooking” or experiencing thermal runaway. Dielectric measurements of renal slices (medulla and cortex) were performed as a function of temperature at a frequency of 918 MHz for a Me₂SO cryoprotectant concentration of 5% (0.7 M). Results of these measurements were then employed as an input to analytical computer models which were used to predict the internal field intensities and power distribution results for both frozen and thawed kidneys. From these predictions, a 918-MHz EM illuminator for thawing canine kidneys was designed and fabricated. Twenty-seven kidneys were thawed using this illumination system. Of these, excellent uniformity of thawing was achieved for 17 kidneys, good uniformity for 8 kidneys, and for only 2 kidneys was thawing uniformity fair to poor.     

Effects of holding temperature and handling stress on the upper thermal tolerance of threadfin shad Dorosoma petenense

The critical thermal maxima (T_MAX) of threadfin shad Dorosoma petenense exposed to standardized stress (30 s handling in a dip‐net), simulating stressors endured during fish loading before transport, were measured over a range of holding temperatures (15, 20 and 25° C). Dorosoma petenense T_MAX showed a significant thermal effect, displaying mean ±s.d . critical thermal maxima of 26·5 ± 1·6, 30·9 ± 1·2 and 33·3 ± 1·4° C, when tested at temperatures of 15, 20 and 25° C, respectively. Dorosoma petenense T_MAX levels were also affected by stress, with handled fish showing significantly lower values than control fish exposed to 15 (mean ±s.d . T_MAX = 25·6 ± 2·0° C), 20 (27·6 ± 2·8° C) and 25° C (32·0 ± 2·6° C). In addition to providing basic information on D. petenense thermal tolerance, experimental results suggest that fishery managers should consider the whole suite of potential stressors, such as air exposure during handling and fish loading, when developing management criteria.     

SEASONAL EFFECT ON THE RELATIONSHIP BETWEEN THE PHOTOSYNTHETIC CAPACITY OF INTERTIDAL MICROPHYTOBENTHOS AND TEMPERATURE1

The response of the photosynthetic capacity (P_max) of microphytobenthos to short-term variations of temperature (in the range 5–35° C) was assessed on a seasonal basis. The relationship is described mathematically, and relevant physiological parameters are identified: P_MAX, the maximum value of P_max achieved at T_opl, the optimum temperature. Estimated values of T_opt do not change significantly throughout the year and remain close to 25° C. It is thus concluded that T_opt is not influenced by seasonal variations in the daily range of mud surface temperature. Identical conclusions hold for T_max (ca. 38° C), the thermal threshold beyond which no photosynthesis occurs. Conversely, P_MA estimates exhibit substantial variability: P_MAX (mean ± root mean square error) is highest in April (11.18 ± 0.42 [μg C · [μg Chl a]^?1· h^?1) during the beginning of the annual increase in temperature, photoperiod, and maximum irradiance and is lowest in December (3.04 ± 0.16 μg C · [μg Chl a]^?1· h^?l). From an ecological point of view, the short-term and seasonal variations of P_MAX suggest that the microphytobenthic community takes advantage of the abiotic spring environmental conditions, allowing the onset of the bloom. Nevertheless, no “acclimation strategy” (i.e. shifts in T_opt and T_max that prevent temperature inhibition in summer or improve photosynthetic rates in winter) is apparent from our results.     

A differential heat-conduction microcalorimeter for heat-capacity measurements of fluids

A heat-conduction calorimeter has been developed for measuring small changes in heat capacity of milligram samples of membrane lipid dispersed in water as a function of temperature. The operation of the instrument is based on the principle that the thermal response of the sample to a short (10 s), electrically generated heat burst is a function of the diffusivity of the sample. Modeling studies of the instrument's performance have revealed that the output response after the heat burst is a function of only the heat capacity, ϱC_p. Calibration of the instrument experimentally confirmed this behavior. This feature obviated the need to measure the thermal conductivity in order to determine ϱC_p from the diffusivity equation, η = γ/ϱC_p. The calorimeter has the following characteristics: reproducibility of loading: ± 400 μJ/C° · cm³; baseline stability: ± 10 μJ/C° · cm³ per 36 h; resolution (± 1 S.D.): ± 50 μJ/C° · cm³; sample size 600 μl.     

Neutron Imaging of Meat during Cooking

Neutron imaging was used to follow the impact of cooking on beef meat. During online cooking, the cartography realised on image collected shows neutron attenuation per zone. Some data points were taken on the edge to highlight higher attenuation variations because of “microscopic” shrinkage of the meat at 70 °C. Some others points were taken in the centre of the sample, which first showed smaller decreases at 75 °C and then an increase around 80 °C. These smaller attenuation variations are possibly linked to denaturation of connective tissue which in turn influenced meat microstructure allowing the release of entrapped water and increase the thickness of the sample.     

Thermal-Stable Proteins of Fruit of Long-Living Sacred Lotus Nelumbo nucifera Gaertn var. China Antique

Single-seeded fruit of the sacred lotus Nelumbo nucifera Gaertn var. China Antique from NE China have been shown to remain viable for as long as ~1,300 years, determined by direct radiocarbon-dating, and to have a germination rate of 84 %. The pericarp, a fruit tissue that encloses the single seeds of Nelumbo, is one of the major factors contributing to fruit longevity. Proteins that are heat stable and have a protective function are equally important to such centuries-long seed viability. We document proteins of Nelumbo fruit that are able to withstand heating, 32 % of which remained soluble in the 110 °C-treated embryo axis of a 549-year-old fruit and 76 % retained fluidity in its cotyledons. The genome of Nelumbo has recently been published and annotated. The amino-acid sequences of 11 “thermal proteins” (soluble at 100 °C) of modern Nelumbo embryo axes and cotyledons, identified by mass spectrometry, Western blot and bioassay, are assembled and aligned with those of an archaeal hyperthermophile Methancaldococcus jannaschii (“Mj,” an anaerobic methanogen having a growth optimum of 85 °C) and with those of five mesophile angiosperms. These thermal proteins have roles in protection and repair under stress. More than half (55 %) of the durable Nelumbo thermal proteins are present in the archaean Mj, indicating their ancient history. One Nelumbo protein-repair enzyme exhibits activity at 100 °C, having a heat-tolerance higher than the comparable enzyme of Arabidopsis. A list of 30 sequenced but unassembled thermal proteins of Nelumbo is appended.     

Upper thermal tolerance of closely related Danio species

The main finding of this study was that measuring maximum heart rate during incremental warming was an effective tool to estimate upper thermal limits in three small cyprinid Danio species, which differed significantly. Arrhenius breakpoint temperature for maximum heart rate, purportedly an index of optimum temperature, was 21·2 ± 0·4, 20·1 ± 0·4 and 18·9 ± 0·8° C (mean ± s.e .) for zebrafish Danio rerio, pearl danio Danio albolineatus and glowlight danio Danio choprae, respectively. The temperature where cardiac arrhythmias were first induced during warming (T_arr) was 36·6 ± 0·7, 36·9 ± 0·8 and 33·2 ± 0·8° C (mean ± s.e .) and critical thermal maximum (T_Cm) was 39·9 ± 0·1, 38·9 ± 0·1 and 37·2 ± 0·1° C (mean ± s.e .) for D. rerio, D. albolineatus and D. choprae, respectively. The finding that T_arr was consistently 3–4° C lower than T_Cm suggests that collapse of the cardiac life support system may be a critical trigger for upper temperature tolerance. The upper thermal limits established here, which correlate well with a broad natural environmental temperature range for D. rerio and a narrow one for D. choprae, suggest that upper thermal tolerance may be a genetic trait even among closely related species acclimated to common temperatures.     

Responses of skilletfish Gobiesox strumosus to high temperature and low oxygen stress

This study quantified physiological responses of skilletfish Gobiesox strumosus exposed to thermal and oxic stress. Fish acclimated at 12, 22 and 32° C had low oxygen tolerance values (mean ±s.d .) of 0·40 ± 0·09, 0·40 ± 0·08 and 0·35 ± 0·03, and critical thermal maxima (mean ±s.d .) of 33·2 ± 0·5, 38·1 ± 0·0 and 39·5 ± 0·3° C, respectively. Furthermore, G. strumosus were oxygen conformers at all acclimation temperatures, i.e. the fish allowed oxygen consumption rates to decrease with ambient oxygen concentration. High temperature tolerance, low oxygen tolerance and decreasing metabolic rates during hypoxic events allow the fish to survive harsh environmental conditions encountered in their natural environment.     

Myelin basic protein in frozen and unfrozen bovine brain: a study of autolytic changes in situ.

Abstract— Frozen and unfrozen bovine brains were used to determine the extent of in situ degradation of myelin basic protein. The following three parameters were investigated. (1) The time interval between death and sampling of the tissue, (2) the effective temperature of the tissue during this interval, and (3) the effect of freezing and thawing on the subsequent autolysis of myelin basic protein. Polyacrylamide gel electrophoresis was carried out on unfrozen white matter solubilized with phenol-formic acid–water. The resulting electrophoretic pattern showed no qualitative changes in the myelin basic protein after tissue incubation at 4° or 23°C for up to 24 h. When myelin basic protein was extracted, purified and quantitated, there was no apparent decrease within 24 h of incubation at 23°C. However, if the tissue was frozen and thawed prior to incubation, there was a rapid disappearance of myelin basic protein such that only 10% remained after 24 h of incubation. Basic protein extracted from frozen or unfrozen tissue that had undergone autolysis for up to 24 h was found to be encephalitogenic in guinea pigs. Electron microscopy of frozen and thawed material showed separation and fraying of myelin lamellae. It is postulated that the above morphological changes probably render the basic protein readily accessible to proteolytic enzymes.     

Physical changes in specimens of five species of Cyprinidae preserved in ethanol and frozen

Preservation in 30% ethanol and freezing to a temperature of ?20 ± 2° C is an appropriate method for measurement of fish eggs, larvae and juveniles. Egg diameter of the common carp Cyprinus carpio increased insignificantly by 1·32% after preservation compared with live size. The total length (L_T) of 1 day post‐hatching (dph) larvae as well as the standard length (LS) of 16 dph larvae of C. carpio increased significantly (2·95 and 1·50%, respectively) after preservation. Egg diameter as well as the L_T of 1 dph larvae of barbel Barbus barbus increased significantly after preservation, by 1·74 and 1·96%, respectively over their original size. The standard length (L_S) of 14 dph larvae of B. barbus as well as juveniles of B. barbus, crucian carp Carassius carassius, common nase Chondrostoma nasus and tench Tinca tinca decreased significantly after preservation (?0·56 to ?5·54%), whereas their body mass increased significantly (11·46–18·57%). Preserved eggs of C. carpio and B. barbus were hard, round and transparent. The larvae and juveniles of examined fishes, preserved in frozen ethanol, were straight, flexible and easily measurable after 60 days. Integrity of body surface and fins, as well as preservation of colours were much better in larvae or juveniles frozen and thawed only once than in specimens frozen and thawed thrice. Cooling in 30% ethanol to a temperature of 6 ± 2° C and freezing in water to a temperature of ?20 ± 2° C are not appropriate preservation methods for eggs and larvae of C. carpio (1 and 16 dph).     

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements

Heat conduction calorimeters are widely used in biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015°C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003°C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box.An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution through the solution in the cell can be the dominant factor in determining the rise-time of the instrument.The heat conduction calorimeter described here has improved characterics: a baseline stability of 200 nJ · s^?1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ · s^?1; a sensitivity of 6.504 ± 0.045 J · V^?1 · s^?1 referred to the sensor output; and a rise-time of 122 s for the 10–90% response.     

The effect of storage temperature and inoculum level on the time of onset of ‘blown pack’ spoilage

Aims: To examine the effect of storage temperature and inoculum level on the time of onset of ‘blown pack’ spoilage (BPS) caused by psychrotolerant bacteria in vacuum‐packed (VP) meats. Methods and Results: Gas‐producing species and strains (n = 11), recovered in our laboratory or reported as associated with BPS, were inoculated onto beef or lamb meat pieces at final levels of <10, 10, 10² and 10³ CFU cm^?2, VP and stored at ?1·5, 1 or 4°C. Six strains produced observable amounts of gas within 42 days and a further four strains produced gas within 100 days. BPS was observed earliest in VP meats inoculated with Clostridium estertheticum ssp. estertheticum at all inoculum levels/storage temperature combinations examined. Storage temperature and inoculum level significantly affected (P < 0·001 and P < 0·05 respectively) the onset of BPS in all cases. Conclusions: Controlling contamination levels and lowering the storage temperature delay the onset of BPS. Significance and Impact of the Study: The study demonstrates the positive effects of low contamination–low temperature as control interventions preventing/delaying BPS in VP chilled meats and identifies some of the contaminants most likely to cause BPS in chilled stored VP meat products.