Toshie Koyama

Ice nucleation in emulsified aqueous solutions of antifreeze protein type III and poly(vinyl alcohol).

Antifreeze protein (AFP) III and poly(vinyl alcohol) (PVA) are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation. However, the effectiveness of these anti-INAs in inhibiting ice nucleation in water-in-oil (W/O) emulsions, in which homogeneous ice nucleation can be experimentally simulated, is unclear. In this study, the ice nucleation temperature in emulsified solutions of AFP III, PVA, and other nonanti-INA polymers was measured, and then the nucleation rate was analyzed based on classical nucleation theory. Results showed that ice nucleation was surface-initiated and, except for PVA solutions, probably caused heterogeneously by the emulsifier, SPAN 65, at the droplet surfaces. In this nucleation mode, AFP III had no significant effect on the ice nucleation rate. In contrast, PVA exhibited ice-nucleating activity only at the droplet surfaces, suggesting that the nucleation is due to the interaction between PVA and SPAN 65.

Inactivation of Ice Nucleating Activity of Silver Iodide by Antifreeze Proteins and Synthetic Polymers

Antifreeze proteins (AFPs) and poly(vinyl alcohol) (PVA) are known as anti-ice nucleating agents (anti-INAs), which inhibit ice nucleation initiated by ice nucleating agents (INAs). Although the effectiveness of anti-INAs depends on the type of INA, most previous studies on anti-INAs used only a few types of biological INAs as targets to inactivate. In this study, the effects of fish AFPs (AFP I and AFP III) and PVA on the ice nucleating activity of silver iodide (AgI) were measured by using emulsified solutions. Results showed that AgI was inactivated not only by AFPs and PVA but also by two other polymers previously not considered as anti-INAs, namely, poly(vinylpyrrolidone) and poly(ethylene glycol). Even in the presence of AgI, a non-negligible fraction, typically more than 10%, of emulsified droplets of these anti-INA solutions at 1.0 mg mL(-1) was supercooled to about -37 °C, which corresponds to ice nucleation temperature measured in the absence of AgI.

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-D-glucopyranoside (K3Glc), kaempferol 7-O-β-D-glucopyranoside (K7Glc) and quercetin 3-O-β-D-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.

Freezing activities of flavonoids in solutions containing different ice nucleators.

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 μL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.

Anti-ice nucleating activity of polyphenol compounds against silver iodide ☆

Freeze-avoiding organisms survive sub-zero temperatures without freezing in several ways, such as removal of ice nucleating agents (INAs), production of polyols, and dehydration. Another way is production of anti-ice nucleating agents (anti-INAs), such as has been reported for several antifreeze proteins (AFPs) and polyphenols, that inhibit ice nucleation by inactivating INAs. In this study, the anti-ice nucleating activity of five polyphenol compounds, including flavonoid and tannin compounds of both biological and synthetic origin, against silver iodide (AgI) was examined by measuring the ice nucleation temperature in emulsified polyphenol solutions containing AgI particles. The emulsified solutions eliminated the influence of contamination by unidentified INAs, thus enabling examination of the anti-ice nucleating activity of the polyphenols against AgI alone. Results showed that all five polyphenol compounds used here have anti-ice nucleating activities that are unique compared with other known anti-INAs, such as fish AFPs (type I and III) and synthetic polymers (poly(vinyl alcohol), poly(vinylpyrrolidone) and poly(ethylene glycol)). All five polyphenols completely inactivated the ice nucleating activity of AgI even at relatively low temperatures, and the first ice nucleation event was observed at temperatures between -14.1 and -19.4°C, compared with between -8.6 and -11.8°C for the fish AFPs and three synthetic polymers. These anti-ice nucleating activities of the polyphenols at such low temperatures are promising properties for practical applications where freezing should be prevented.

O-Aryl-Glycoside Ice Recrystallization Inhibitors as Novel Cryoprotectants: A Structure–Function Study

Low-molecular-weight ice recrystallization inhibitors (IRIs) are ideal cryoprotectants that control the growth of ice and mitigate cell damage during freezing. Herein, we describe a detailed study correlating the ice recrystallization inhibition activity and the cryopreservation ability with the structure of O-aryl-glycosides. Many effective IRIs are efficient cryoadditives for the freezing of red blood cells (RBCs). One effective cryoadditive did not inhibit ice recrystallization but instead inhibited ice nucleation, demonstrating the significance of inhibiting both processes and illustrating the importance of this emerging class of cryoprotectants.

Anti-Ice Nucleating Activity of Surfactants Against Silver Iodide in Water-in-Oil Emulsions

Various water-soluble substances are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation initiated by ice nucleating agents (INAs). Among them, several surfactants are reportedly effective as anti-INAs especially against silver iodide (AgI), which is a typical inorganic INA that induces heterogeneous ice nucleation at relatively high temperatures. In this study, the anti-ice nucleating activities of seven surfactants were examined in emulsified surfactant solutions containing AgI particles. Among previously reported anti-INAs (e.g., antifreeze proteins (AFPs), polyphenol compounds and synthetic polymers), a cationic surfactant used in this study, hexadecyltrimethylammonium bromide (C16TAB), showed the highest anti-ice nucleating activity against AgI. Based on the unique concentration-dependent dispersibility of AgI particles in C16TAB solution, the anti-ice nucleating activity of C16TAB must be caused by the adsorption of C16TAB molecules on AgI surfaces either as a monolayer or a bilayer depending on the C16TAB concentration.

Culture of Porcine Hepatocytes Using Radial Flow Bioreactor System

Porcine primary hepatocytes were cultured in a radial flow bioreactor system to examine the effects of ascorbic acid 2-phosphate (Asc2-P) and dissolved oxygen concentration (DO) on the ammonium metabolism. The hepatocytes were isolated from the liver of slaughtered pig, and 2 × 108 hepatocytes were inoculated into the bioreactor. At 6.86 mg/l DO, ammonium metabolic rate of hepatocytes cultured with 1.5 mmol/l Asc2-P was higher than that without Asc2-P, and was maintained over 10 days. At 10.0 mg/l DO, the activity was enhanced and maintained over 1 month. These results show that the hepatocytes can maintain the function for long period in the bioreactor with Asc2-P at 10.0 mg/l DO.