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145. Screening and functional analyses of ice crystal structure-controlling materials from antarctic fungi
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Abstracts / Cryobiology 53 (2006) 367–446
throughout the entire cytoplasm. The most distinct observed ultrastructural feature was the distribution of small oil droplets (approximately 200–500 nm in diameter) just beneath the plasma membranes. After equilibrium freezing to 10 °C, the cells are shrunken by extracellular freezing, but the ER in these boreal softwoods maintained their cylindrical form without essential redistribution of ER. Rather, small oil droplets very closely covered the sub-surface of plasma membranes. We showed that ultrastructural changes in cortical parenchymal cells in boreal softwood species under equilibrium freezing clearly differed from those of the cold hardy boreal hardwood species. We speculate the small oil droplets beneath the plasma membrane might play a role in protecting cortical parenchyma cells from equilibrium freeze-induced injury, instead of formation of multiplex lamellar of ER in boreal hardwood species. (Conflict of interest: None declared. Source of funding: Grant-in Aid for Fundamental Research from the Ministry of Education, Science, Sports and Culture, Japan.) doi:10.1016/j.cryobiol.2006.10.144
144. Purification and characterization of antifreeze protein from a mushroom, Flammulina velutipes. Hidehisa Kawahara, Kazutaka Kawakami, Hitoshi Obata, Biotechnology, Kansai University, 564-8680 Suita, Japan Antifreeze proteins (AFPs) are a group of ice-binding proteins that inhibit the growth of ice. They have thermal hysteresis (TH) activity, which is the non-colligative depression of the freezing point, and ice recrystallization-inhibiting activity in the frozen state over 10 °C. AFPs have been found in fishes, plants, various and microorganisms including fungi, lichen and bacteria. The microorganisms that are capable of producing AFPs cannot be used in the food industry. We have screened AFP-producing mushrooms, which are commercial mushrooms in Japan. We found that a mushroom, Flammulina velutipes, which is known as Enokitake, can produce AFP in the culture broth. F. velutipes was cultured for 1 week at 18 °C and then for 1 week at 4 °C in the YG medium containing vitamin B1 (pH 5.66). The AFP was purified by DEAE–Toyopearl 650 M, Superdex 75 and Native PAGE. The molecular weight of the purified AFP was about 60 kDa as a monomer by SDS–PAGE and gel filtration chromatography. The ice crystal shape with low concentrations of AFP was dendritic with hexagonal symmetry, and the ice crystal shape with high concentrations of AFP was a hexagonal bipyramid, similar to fish AFP. The TH value was 0.08 °C constant over the concentration 0.25 mg/ml. Also, this AFP was sensitive to treatment by heat. The TH activity was decreased by heat treatment at >25 °C for 30 min. The commercial use of this AFP for various frozen foods will be possible in the future after solving the low level of thermal stability. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.145
145. Screening and functional analyses of ice crystal structurecontrolling materials from antarctic fungi. Tomonobu Takemura, Hidehisa Kawahara, Hitoshi Obata, Biotechnology, Kansai University, 564-8680 Suita-shi, Japan
Ice crystal structure-controlling materials (ICSCs) are a group of ice-crystal binding materials that can inhibit the growth and structure of ice crystals. They have thermal hysteresis (TH) activity, which is the non-colligative depression of the freezing point by inhibiting the growth of ice crystal. ICSCs have the ability to modify the ice crystal structure, like the hexagonal ice crystal growth in the presence of ICSCs. Almost antifreeze protein have a high level of ice recrystallization-inhibiting activity in the frozen state over 10 °C. However, ICSC have a low level of ice recrystallization-inhibiting activity. In this study, we screened Antarctic fungi, which can produce various ICSCs, and analyzed various functions of ICSC. Forty strains of fungi isolated from some soils that originated from Antarctica, were cultivated in three media (LB : Difco, NB : Difco, TSB : Bacto) for 2 weeks at 18 °C and each culture broth was measured for TH activity. Among 40 strains of Antarctic fungi, only seven strains exhibited TH activity (about 0.02–0.06 °C). These seven strains belonged to the genus Fusarium sp., Oidodendron sp., Cladosporium sp., Phoma sp. and Penicillium sp. As Penicillium bilaiae No. 205 may be useful in the industry and had high TH activity, so this strain was selected to produce ICSC. The optimum medium to produce ICSC in this strain was the Nutrient Broth. All AFPs were expressed under cold condition, but this strain could produce ICSC in the culture broth at 30 °C. We are now working to identify the active materials, to characterize molecular components and to purify ICSC from P. bilaiae No. 205. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.146
146. Acid adaptation improves cryotolerance of Lactobacillus bulgaricus CFL1. Fernanda Streit a, Georges Corrieu a, Catherine Beal b, a Institut National de la Recherche Agronomique, UMR GMPA, 78850 Thiverval-Grignon, France; b Institut National Agronomique Paris-Grignon, UMR GMPA, 78850 ThivervalGrignon, France Stabilization of micro-organisms, specially freezing, generally affects the survival and the physiological state of the cells. However, a preliminary adaptation of the bacteria enables them to better resist these operations, thus leading to strains with optimal properties for their utilization. This is particularly interesting for bacteria that are used for manufacturing fermented food, such as lactic acid bacteria. This work aims to study the effect of an acid adaptation on the cryotolerance of the lactic bacteria Lactobacillus bulgaricus CFL1. The acid adaptation was carried out, at the end of the fermentation, by applying different pH conditions (4.5–6.0) during different times (0–60 min). Analysis of variance showed that pH and duration of acidification had significant effects on all the parameters that characterize the metabolic activity of the cells—initial acidification activity and loss of acidification activity during freezing and frozen storage at 20 °C. We found that an acid adaptation at pH 5.15 for 30 min allowed the bacteria to better resist freezing and frozen storage. In the future, the biological mechanisms that induce this adaptation will be elucidated, by comparing the membrane fatty acid composition and the proteome of adapted and non-adapted cells. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.147
Abstracts / Cryobiology 53 (2006) 367–446
throughout the entire cytoplasm. The most distinct observed ultrastructural feature was the distribution of small oil droplets (approximately 200–500 nm in diameter) just beneath the plasma membranes. After equilibrium freezing to 10 °C, the cells are shrunken by extracellular freezing, but the ER in these boreal softwoods maintained their cylindrical form without essential redistribution of ER. Rather, small oil droplets very closely covered the sub-surface of plasma membranes. We showed that ultrastructural changes in cortical parenchymal cells in boreal softwood species under equilibrium freezing clearly differed from those of the cold hardy boreal hardwood species. We speculate the small oil droplets beneath the plasma membrane might play a role in protecting cortical parenchyma cells from equilibrium freeze-induced injury, instead of formation of multiplex lamellar of ER in boreal hardwood species. (Conflict of interest: None declared. Source of funding: Grant-in Aid for Fundamental Research from the Ministry of Education, Science, Sports and Culture, Japan.) doi:10.1016/j.cryobiol.2006.10.144
144. Purification and characterization of antifreeze protein from a mushroom, Flammulina velutipes. Hidehisa Kawahara, Kazutaka Kawakami, Hitoshi Obata, Biotechnology, Kansai University, 564-8680 Suita, Japan Antifreeze proteins (AFPs) are a group of ice-binding proteins that inhibit the growth of ice. They have thermal hysteresis (TH) activity, which is the non-colligative depression of the freezing point, and ice recrystallization-inhibiting activity in the frozen state over 10 °C. AFPs have been found in fishes, plants, various and microorganisms including fungi, lichen and bacteria. The microorganisms that are capable of producing AFPs cannot be used in the food industry. We have screened AFP-producing mushrooms, which are commercial mushrooms in Japan. We found that a mushroom, Flammulina velutipes, which is known as Enokitake, can produce AFP in the culture broth. F. velutipes was cultured for 1 week at 18 °C and then for 1 week at 4 °C in the YG medium containing vitamin B1 (pH 5.66). The AFP was purified by DEAE–Toyopearl 650 M, Superdex 75 and Native PAGE. The molecular weight of the purified AFP was about 60 kDa as a monomer by SDS–PAGE and gel filtration chromatography. The ice crystal shape with low concentrations of AFP was dendritic with hexagonal symmetry, and the ice crystal shape with high concentrations of AFP was a hexagonal bipyramid, similar to fish AFP. The TH value was 0.08 °C constant over the concentration 0.25 mg/ml. Also, this AFP was sensitive to treatment by heat. The TH activity was decreased by heat treatment at >25 °C for 30 min. The commercial use of this AFP for various frozen foods will be possible in the future after solving the low level of thermal stability. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.145
145. Screening and functional analyses of ice crystal structurecontrolling materials from antarctic fungi. Tomonobu Takemura, Hidehisa Kawahara, Hitoshi Obata, Biotechnology, Kansai University, 564-8680 Suita-shi, Japan
Ice crystal structure-controlling materials (ICSCs) are a group of ice-crystal binding materials that can inhibit the growth and structure of ice crystals. They have thermal hysteresis (TH) activity, which is the non-colligative depression of the freezing point by inhibiting the growth of ice crystal. ICSCs have the ability to modify the ice crystal structure, like the hexagonal ice crystal growth in the presence of ICSCs. Almost antifreeze protein have a high level of ice recrystallization-inhibiting activity in the frozen state over 10 °C. However, ICSC have a low level of ice recrystallization-inhibiting activity. In this study, we screened Antarctic fungi, which can produce various ICSCs, and analyzed various functions of ICSC. Forty strains of fungi isolated from some soils that originated from Antarctica, were cultivated in three media (LB : Difco, NB : Difco, TSB : Bacto) for 2 weeks at 18 °C and each culture broth was measured for TH activity. Among 40 strains of Antarctic fungi, only seven strains exhibited TH activity (about 0.02–0.06 °C). These seven strains belonged to the genus Fusarium sp., Oidodendron sp., Cladosporium sp., Phoma sp. and Penicillium sp. As Penicillium bilaiae No. 205 may be useful in the industry and had high TH activity, so this strain was selected to produce ICSC. The optimum medium to produce ICSC in this strain was the Nutrient Broth. All AFPs were expressed under cold condition, but this strain could produce ICSC in the culture broth at 30 °C. We are now working to identify the active materials, to characterize molecular components and to purify ICSC from P. bilaiae No. 205. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.146
146. Acid adaptation improves cryotolerance of Lactobacillus bulgaricus CFL1. Fernanda Streit a, Georges Corrieu a, Catherine Beal b, a Institut National de la Recherche Agronomique, UMR GMPA, 78850 Thiverval-Grignon, France; b Institut National Agronomique Paris-Grignon, UMR GMPA, 78850 ThivervalGrignon, France Stabilization of micro-organisms, specially freezing, generally affects the survival and the physiological state of the cells. However, a preliminary adaptation of the bacteria enables them to better resist these operations, thus leading to strains with optimal properties for their utilization. This is particularly interesting for bacteria that are used for manufacturing fermented food, such as lactic acid bacteria. This work aims to study the effect of an acid adaptation on the cryotolerance of the lactic bacteria Lactobacillus bulgaricus CFL1. The acid adaptation was carried out, at the end of the fermentation, by applying different pH conditions (4.5–6.0) during different times (0–60 min). Analysis of variance showed that pH and duration of acidification had significant effects on all the parameters that characterize the metabolic activity of the cells—initial acidification activity and loss of acidification activity during freezing and frozen storage at 20 °C. We found that an acid adaptation at pH 5.15 for 30 min allowed the bacteria to better resist freezing and frozen storage. In the future, the biological mechanisms that induce this adaptation will be elucidated, by comparing the membrane fatty acid composition and the proteome of adapted and non-adapted cells. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.147