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36. Glass transition and chemical stability of model freeze-dried foods
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Abstracts / Cryobiology 59 (2009) 370–418
34. Effect of sugar on the osmotic injury of PC-3 cells in hypertonic NaCl solutions. Takuro Yoshimura, *Hiroshi Takamatsu, Department of Mechanical Engineering, Kyushu University, Fukuoka, Japan Osmotic dehydration due to elevated concentration of electrolytes is believed to be one of the major causes of cell injury during freezing. Our previous study has shown that the electrolyte concentration had more effect on cell damage than the mechanical stress from the extracellular ice at lower freezing temperatures [1]. We have also demonstrated that the progress in cell damage was biphasic during the osmotic challenge similar to that during a freeze–thaw process; the viability decreased first after the increase in extracellular NaCl concentration due to dehydration and then after return to the isotonic condition due to rehydration [2]. Rehydration was substantially more responsible for cell injury than dehydration. The fact that the post-hypertonic viability decreased with the time of exposure to hypertonic solution suggested that the rehydration-induced injury was a consequence of timedependent alteration of the plasma membrane. The objective of the present study was to examine the effect of sugar on the osmotic injury of cells. Sucrose and trehalose were tested as additives to NaCl solutions because of their potential as cryoprotective agents. Human prostate cancer cells (PC-3 cells) that were suspended in NaCl solutions with and without additives were exposed to hyperosmotic conditions. The osmolality was increased from 0.4 osmol/kg to 8.6 kg/osmol at 2.2 osmol/min in average, and decreased at the same rate to the initial condition. The basic solutions were 0.21 M NaCl solution, 0.15 M NaCl solution with 0.05 M sucrose, and 0.15 M NaCl solution with 0.05 M trehalose. The concentration ratio between sugars and NaCl was kept constant during the increase in the concentration. Cells were exposed to the highest osmolality for 10 or 30 min. The cell survival was measured with PI that had been mixed in the solutions at 15 mM. All experiments were conducted at 23 °C. Cell survival after exposure to hyperosmotic solutions for 10 min was approximately 60% and no significant difference was found between that in different solutions. However, in longer time of exposure for 30 min, the survival in the solution with sucrose and trehalose was significantly higher than that in the NaCl solution, which decreased to approximately 20%. No significant difference was observed between sucrose and trehalose. The occurrence of cell damage in solutions with sucrose or trehalose was biphasic as has been observed in our previous study with NaCl solutions [2]. However, addition of sucrose or trehalose reduced the rehydration-induced damage after cell swelling. This suggested that the sugar had a potential to protect plasma membrane from alteration due to close apposition of membranes. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 18360104).)
36. Glass transition and chemical stability of model freeze-dried foods. *Kiyoshi Kawai a, Paveena Srirangsan b, Kaori Tsuji b, Toru Suzuki b, a Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan, b Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Most of dry foods are in the amorphous state, at least partially, and thus turn into the glassy state at temperatures below the glass transition temperature (Tg). It is assumed that glassy dry foods are stable chemically and physically because their molecular mobility is extremely low. In addition, it is expected that the higher the (Tg), the lower the molecular mobility and the better storage stability is at a reference temperature, because (Tg) means the temperature at which the viscosity of amorphous materials becomes constant at approximately 1012 Pa s. The non-enzymatic browning reaction (NBR) is a typical chemical reaction observed in dry foods. The relationship between (Tg) and NBR rate of glassy dry foods, however, is not completely understood. In order to obtain some insight into the subject, various model freeze-dried glassy foods were prepared, and the NBR rate was compared. The mixtures of amino acid (e.g., lysine and glycine), reducing saccharide (e.g., glucose and maltose), and polyvinylpyrrolidone (PVP) of varying molecular weight were employed as model freeze-dried food systems. The samples were stored at 40–70 °C, and the extent of NBR was investigated spectrophotometrically. The value of (Tg) was investigated by using DSC. The (Tg) of the samples depended strongly on the molecular weight of PVP and residual moisture. In comparison of their NBR rates, it was confirmed that the NBR rate decreased with increase in (Tg). The results suggest that the molecular mobility of freeze-dried glassy food systems is an important factor for the progress of NBR. On the other hand, there was a minor effect of types of amino acid and reducing saccharide on the (Tg) value. Their NBR rate, however, strongly depended on the types of amino acid and reducing saccharide. The results suggest that NBR progress partially with independent of the molecular mobility. In previous studies, it is stated that NBR reactants (amino acid and saccharide) form hydrogen bonds in the dehydrated state. The hydrogen-bonding NBR reactants will react readily without diffusion in the glassy system. From these observations, it is thought that the NBR rate of freeze-dried glassy food systems is affected not only by molecular mobility, but also by hydrogen-bond formation in the dehydrated state. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2009.10.050
References Principles of cryopreservation [1] Takamatsu H, Zawlodzka S. Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions. Cryobiology 2006;53:1–11. [2] Zawlodzka S, Takamatsu H. Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperature above 0 °C. Cryobiology 2005;50:58–70. doi:10.1016/j.cryobiol.2009.10.048
35. Effects of pre-cooling rates on the freeze-dried pig artery: An analysis concerning the sublimation interface. Meng-fang Liu, Le-ren Tao, Jian-qing Wu, Shu-hong Zhang, Yong-fu Li, Institute of Cryogenic Technology and Food Freezing, University of Shanghai for Science and Technology, Shanghai 200093, China More and more researches indicate that transplantation of vessels that have been preserved by vacuum freeze-drying is feasible. When rehydrated, freeze-dried pig arteries are transplanted the differences between the rehydrated and the fresh are not remarkable. However analysis by Micro-CT, can scan the inner structure of samples nondestructively and freeze-dried samples that had been cooled more rapidly had a higher porosity and gray value (a lighter color) after rehydration. In this experiment, the cooling rates were 0.5, 1 and 2 K/min, respectively. The experimental conditions were: the temperatures of freeze-drying was set at 70, 20 and 15 °C, respectively, for pre-freezing, primary drying and the secondary drying. The thickness of the walls thinned gradually and the obvious change of the sublimation interface occurred mainly in the secondary drying phase. This experiment, analyzing the process of freeze-drying in the pig artery, verified some theories of crystallization, heat and mass transfer analysis. But it is a basic experiment and should rely on many aspects such as physical properties, mechanical properties, immunology, transplantation, etc., in vascular transplantation. Thanks for Jianqing WU’s help with the performance test. (Conflicts of interest: None declared. Source of funding: Shanghai Leading Academic Discipline Project, Project Number: S30503.) doi:10.1016/j.cryobiol.2009.10.049
37. Computational predictions of the cryopreservation of human oocytes. *J.J. McGrath, S.A. Unhale, Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA Computer models capable of predicting the responses of human oocytes to the chemical and thermal processing involved in cryopreservation have enormous potential. In principle, such models can be used to design optimal protocols based on basic principles. The potential benefits include improved recovery, faster processing and lower cost – all based on a fundamental understanding rather than empiricism. Most of the major steps defined in a published, slow cooling protocol for cryopreserving human oocytes in solutions containing 1,2-propanediol and sucrose have been modeled [Fabri et al., Hum. Reprod., 16 (2001) 411]. One of the strengths of the approach taken is that the individual steps in the overall cryopreservation process are coupled together in series. In this manner, it is possible to understand the influence of each step in the overall process. Predictions of the multiple steps involved in the addition and removal of permeable and impermeable cryoprotectants suggest that osmotic excursions as well as the concentrations of impermeable and permeable species do not exceed tolerable limits. Predictions of the response of human oocytes to cooling are based on membrane parameters that are known at temperatures above 0 °C. Extrapolation to sub-zero temperatures as implemented here represents a common limitation of this type of modeling. In addition, the ice nucleation parameters required for predicting the intracellular ice formation are not known for human oocytes; nor is the viscosity model for 1,2-propanediol. In the absence of this information, published values for the ice nucleation parameters for dimethyl sulfoxide Me2SO in mouse oocytes as well as the intracellular viscosity model for Me2SO were used [Karlsson et al., Hum. Reprod. 11 (1996) 1296]. The model was ‘‘tuned” to match the experimental cryomicroscopy results for intracellular ice formation (IIF) in human oocytes published previously [Trad et al., Hum. Reprod. 14 (1998) 1569]. With this ‘‘tuned” model it proved possible to predict all of the major results found empirically. In particular, this included the effect of cooling rate on IIF, the temperature at which intracellular ice forms and the beneficial influence of adding sucrose to the cryoprotectant solution. The model predictions for cryoprotectant addition and removal suggested that the published protocol could be simplified considerably without jeopardizing osmotic excursions or solute concentrations. The model predicted that the number of steps could be reduced and the total
Abstracts / Cryobiology 59 (2009) 370–418
34. Effect of sugar on the osmotic injury of PC-3 cells in hypertonic NaCl solutions. Takuro Yoshimura, *Hiroshi Takamatsu, Department of Mechanical Engineering, Kyushu University, Fukuoka, Japan Osmotic dehydration due to elevated concentration of electrolytes is believed to be one of the major causes of cell injury during freezing. Our previous study has shown that the electrolyte concentration had more effect on cell damage than the mechanical stress from the extracellular ice at lower freezing temperatures [1]. We have also demonstrated that the progress in cell damage was biphasic during the osmotic challenge similar to that during a freeze–thaw process; the viability decreased first after the increase in extracellular NaCl concentration due to dehydration and then after return to the isotonic condition due to rehydration [2]. Rehydration was substantially more responsible for cell injury than dehydration. The fact that the post-hypertonic viability decreased with the time of exposure to hypertonic solution suggested that the rehydration-induced injury was a consequence of timedependent alteration of the plasma membrane. The objective of the present study was to examine the effect of sugar on the osmotic injury of cells. Sucrose and trehalose were tested as additives to NaCl solutions because of their potential as cryoprotective agents. Human prostate cancer cells (PC-3 cells) that were suspended in NaCl solutions with and without additives were exposed to hyperosmotic conditions. The osmolality was increased from 0.4 osmol/kg to 8.6 kg/osmol at 2.2 osmol/min in average, and decreased at the same rate to the initial condition. The basic solutions were 0.21 M NaCl solution, 0.15 M NaCl solution with 0.05 M sucrose, and 0.15 M NaCl solution with 0.05 M trehalose. The concentration ratio between sugars and NaCl was kept constant during the increase in the concentration. Cells were exposed to the highest osmolality for 10 or 30 min. The cell survival was measured with PI that had been mixed in the solutions at 15 mM. All experiments were conducted at 23 °C. Cell survival after exposure to hyperosmotic solutions for 10 min was approximately 60% and no significant difference was found between that in different solutions. However, in longer time of exposure for 30 min, the survival in the solution with sucrose and trehalose was significantly higher than that in the NaCl solution, which decreased to approximately 20%. No significant difference was observed between sucrose and trehalose. The occurrence of cell damage in solutions with sucrose or trehalose was biphasic as has been observed in our previous study with NaCl solutions [2]. However, addition of sucrose or trehalose reduced the rehydration-induced damage after cell swelling. This suggested that the sugar had a potential to protect plasma membrane from alteration due to close apposition of membranes. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 18360104).)
36. Glass transition and chemical stability of model freeze-dried foods. *Kiyoshi Kawai a, Paveena Srirangsan b, Kaori Tsuji b, Toru Suzuki b, a Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan, b Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Most of dry foods are in the amorphous state, at least partially, and thus turn into the glassy state at temperatures below the glass transition temperature (Tg). It is assumed that glassy dry foods are stable chemically and physically because their molecular mobility is extremely low. In addition, it is expected that the higher the (Tg), the lower the molecular mobility and the better storage stability is at a reference temperature, because (Tg) means the temperature at which the viscosity of amorphous materials becomes constant at approximately 1012 Pa s. The non-enzymatic browning reaction (NBR) is a typical chemical reaction observed in dry foods. The relationship between (Tg) and NBR rate of glassy dry foods, however, is not completely understood. In order to obtain some insight into the subject, various model freeze-dried glassy foods were prepared, and the NBR rate was compared. The mixtures of amino acid (e.g., lysine and glycine), reducing saccharide (e.g., glucose and maltose), and polyvinylpyrrolidone (PVP) of varying molecular weight were employed as model freeze-dried food systems. The samples were stored at 40–70 °C, and the extent of NBR was investigated spectrophotometrically. The value of (Tg) was investigated by using DSC. The (Tg) of the samples depended strongly on the molecular weight of PVP and residual moisture. In comparison of their NBR rates, it was confirmed that the NBR rate decreased with increase in (Tg). The results suggest that the molecular mobility of freeze-dried glassy food systems is an important factor for the progress of NBR. On the other hand, there was a minor effect of types of amino acid and reducing saccharide on the (Tg) value. Their NBR rate, however, strongly depended on the types of amino acid and reducing saccharide. The results suggest that NBR progress partially with independent of the molecular mobility. In previous studies, it is stated that NBR reactants (amino acid and saccharide) form hydrogen bonds in the dehydrated state. The hydrogen-bonding NBR reactants will react readily without diffusion in the glassy system. From these observations, it is thought that the NBR rate of freeze-dried glassy food systems is affected not only by molecular mobility, but also by hydrogen-bond formation in the dehydrated state. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2009.10.050
References Principles of cryopreservation [1] Takamatsu H, Zawlodzka S. Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions. Cryobiology 2006;53:1–11. [2] Zawlodzka S, Takamatsu H. Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperature above 0 °C. Cryobiology 2005;50:58–70. doi:10.1016/j.cryobiol.2009.10.048
35. Effects of pre-cooling rates on the freeze-dried pig artery: An analysis concerning the sublimation interface. Meng-fang Liu, Le-ren Tao, Jian-qing Wu, Shu-hong Zhang, Yong-fu Li, Institute of Cryogenic Technology and Food Freezing, University of Shanghai for Science and Technology, Shanghai 200093, China More and more researches indicate that transplantation of vessels that have been preserved by vacuum freeze-drying is feasible. When rehydrated, freeze-dried pig arteries are transplanted the differences between the rehydrated and the fresh are not remarkable. However analysis by Micro-CT, can scan the inner structure of samples nondestructively and freeze-dried samples that had been cooled more rapidly had a higher porosity and gray value (a lighter color) after rehydration. In this experiment, the cooling rates were 0.5, 1 and 2 K/min, respectively. The experimental conditions were: the temperatures of freeze-drying was set at 70, 20 and 15 °C, respectively, for pre-freezing, primary drying and the secondary drying. The thickness of the walls thinned gradually and the obvious change of the sublimation interface occurred mainly in the secondary drying phase. This experiment, analyzing the process of freeze-drying in the pig artery, verified some theories of crystallization, heat and mass transfer analysis. But it is a basic experiment and should rely on many aspects such as physical properties, mechanical properties, immunology, transplantation, etc., in vascular transplantation. Thanks for Jianqing WU’s help with the performance test. (Conflicts of interest: None declared. Source of funding: Shanghai Leading Academic Discipline Project, Project Number: S30503.) doi:10.1016/j.cryobiol.2009.10.049
37. Computational predictions of the cryopreservation of human oocytes. *J.J. McGrath, S.A. Unhale, Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA Computer models capable of predicting the responses of human oocytes to the chemical and thermal processing involved in cryopreservation have enormous potential. In principle, such models can be used to design optimal protocols based on basic principles. The potential benefits include improved recovery, faster processing and lower cost – all based on a fundamental understanding rather than empiricism. Most of the major steps defined in a published, slow cooling protocol for cryopreserving human oocytes in solutions containing 1,2-propanediol and sucrose have been modeled [Fabri et al., Hum. Reprod., 16 (2001) 411]. One of the strengths of the approach taken is that the individual steps in the overall cryopreservation process are coupled together in series. In this manner, it is possible to understand the influence of each step in the overall process. Predictions of the multiple steps involved in the addition and removal of permeable and impermeable cryoprotectants suggest that osmotic excursions as well as the concentrations of impermeable and permeable species do not exceed tolerable limits. Predictions of the response of human oocytes to cooling are based on membrane parameters that are known at temperatures above 0 °C. Extrapolation to sub-zero temperatures as implemented here represents a common limitation of this type of modeling. In addition, the ice nucleation parameters required for predicting the intracellular ice formation are not known for human oocytes; nor is the viscosity model for 1,2-propanediol. In the absence of this information, published values for the ice nucleation parameters for dimethyl sulfoxide Me2SO in mouse oocytes as well as the intracellular viscosity model for Me2SO were used [Karlsson et al., Hum. Reprod. 11 (1996) 1296]. The model was ‘‘tuned” to match the experimental cryomicroscopy results for intracellular ice formation (IIF) in human oocytes published previously [Trad et al., Hum. Reprod. 14 (1998) 1569]. With this ‘‘tuned” model it proved possible to predict all of the major results found empirically. In particular, this included the effect of cooling rate on IIF, the temperature at which intracellular ice forms and the beneficial influence of adding sucrose to the cryoprotectant solution. The model predictions for cryoprotectant addition and removal suggested that the published protocol could be simplified considerably without jeopardizing osmotic excursions or solute concentrations. The model predicted that the number of steps could be reduced and the total