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Pilar Bermejobarrera - One of the best experts on this subject based on the ideXlab platform.
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study of cooking on the bioavailability of as co cr cu fe ni se and zn from edible Seaweed
Microchemical Journal, 2013Co-Authors: Cristina Garciasartal, Antonio Moredapineiro, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract This study examines the effects of cooking on the bioavailability of As, Co, Cr, Cu, Fe, Ni, Se and Zn from edible Seaweed. Four edible Galician Seaweeds (Kombu, Wakame, Sea Lettuce and Nori) were cooked following the manufacturer's recommendations. Cr, Fe, Co, Ni, Cu, Zn, As and Se were analyzed in raw and cooked Seaweeds after subjecting them to an acid digestion. The metals were released into the cooking water during the heat treatment with the exception of Ni and Zn in Kombu, Cr, Fe and Co in Wakame, and Zn in Nori, for which almost 100% was retained in the Seaweed. An in vitro digestion method was developed to assess the bioavailability percentages of those metals in the cooked Seaweed. Results suggested that Fe, Zn and Cu are not bioavailable for body functions in the studied matrices. Low dialyzability percentages were found for Cr, Co, Ni, As and Se, ranging from 1.0–16.0%, 3.4–27%, 8.2–12.9%, 9.7–21.0%, and 14.6–61.3%, respectively. Kombu Seaweed was used as a model to perform a mass balance study for evaluating the accuracy of the bioavailability study of cooked Seaweed. Standard deviation homogeneity was corroborated by means of the Cochran's and Bartlett's statistical tests (p-values > 0.05). An ANOVA test was used to compare the mean metal concentrations between and within groups. p-Values higher than 0.05 showed that there were no statistical differences, and the mass balance was adequate. Finally, the influence of the main food constituents (fat, protein, dietary fiber and carbohydrate) on the metal bioavailability was evaluated. Correlation matrix and multivariate analysis based on multiple linear regression indicated positive and negative correlations between the type of Seaweed and metal concentration.
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two dimensional hplc coupled to icp ms and electrospray ionisation esi ms ms for investigating the bioavailability in vitro of arsenic species from edible Seaweed
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Cristina Garciasartal, Pilar Bermejobarrera, Sutthinun Taebunpakul, Emma Stokes, M C Barcielaalonso, Heidi GoenagainfanteAbstract:Edible Seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible Seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible Seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four Seaweed namely kombu, wakame, nori and Sea Lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1 h sonication in a water bath followed by HPLC-ICP-MS resulted in 40-61% of the total As to be found in the buffered aqueous extract, of which 86-110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was Sea Lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in Sea Lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11-16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93-120% and 41% of the dialyzable As fraction for kombu, wakame, nori and Sea Lettuce, respectively. Moreover, the relative As species distribution in Seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in Seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in Sea Lettuce samples was also confirmed.
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use of an in vitro digestion method to evaluate the bioaccessibility of arsenic in edible Seaweed by inductively coupled plasma mass spectrometry
Microchemical Journal, 2011Co-Authors: Cristina Garciasartal, Raquel Dominguezgonzalez, Antonio Moredapineiro, M C Barcielaalonso, Vanessa Romarishortas, Pilar BermejobarreraAbstract:Abstract Different types of edible Seaweeds (Kombu, Wakame, Nori and Sea Lettuce) harvested in the Galician coast (Northwestern Spain) were analyzed for total arsenic quantification before and after being cooked following manufacturer's instructions. Furthermore, the total arsenic in the bioaccessible fraction obtained after simulating a human digestion by an in vitro process was determined in those raw and cooked Seaweeds. The detection of the target element was performed by inductively coupled plasma-mass spectrometry (ICP-MS) which was equipped with a collision cell to avoid polyatomic interferences. Piperazine-NN-bis (2-ethane-sulfonic acid) disodium (PIPES) buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut-off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a BCR-279 certified reference material. The accuracy of the in vitro procedures was established by a mass balance study for Nori and Sea Lettuce which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). Results showed that the effect of cooking Seaweed causes the removal of the element into the cooking water. Dialyzability percentages found in raw Seaweed samples were comparable to those found in cooked Seaweed, except in the case of Sea Lettuce sample for which a lower dialyzability percentage was found when it was cooked.
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bioavailability study using an in vitro method of iodine and bromine in edible Seaweed
Food Chemistry, 2011Co-Authors: Vanessa Romaris Hortas, Raquel Dominguezgonzalez, Antonio Moredapineiro, Cristina Garciasartal, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract Raw edible Seaweed harvested in the Galician coast (Northwester Spain), including two red Seaweed types (Dulse and Nori), three brown Seaweed (Kombu, Wakame and Sea Spaghetti), one green Seaweed (Sea Lettuce) and one microalgae ( Spirulina platensis ) were analysed for total iodine and total bromine, as well as for iodine and bromine bioavailability by in-vitro methods (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked Seaweed sample (canned in brine) consisting of a mixture of two brown Seaweed (Sea Spaghetti and Furbelows) and a derived product (agar–agar) from the red Seaweed Gelidiumm sesquipedale , were also included in the study. All measurements were carried out by inductively coupled plasma–mass spectrometry using tellurium and yttrium as internal standards for iodine and bromine, respectively. An optimised microwave assisted alkaline (TMAH) digestion procedure was used as sample pre-treatment for total iodine and bromine determinations, as well as for the determination of both elements in the non-dialyzable fractions. PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for the intestinal digestion. Accuracy of the method (total bromine and iodine determinations) was assessed by analysing a NIES-09 certified reference material. The accuracy of the in-vitro procedure was established by a mass-balance study which led, after statistical evaluation (95% confidence interval), good accuracy of the whole in-vitro process. The highest dialyzability bromine percentages (36 ± 0.7% and 47 ± 3.0%) were obtained for red Seaweed (Dulse and Nori), while higher dialyzability iodine was assessed for the brown Seaweed (Kombu), around 17% ± 0.7%.
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development of a new sample pre treatment procedure based on pressurized liquid extraction for the determination of metals in edible Seaweed
Analytica Chimica Acta, 2007Co-Authors: Jorge Moredapineiro, Antonio Moredapineiro, Elia Alonsorodriguez, P Lopezmahia, Soledad Muniateguilorenzo, D Pradarodriguez, Pilar BermejobarreraAbstract:A new, simple, fast and automated method based on acetic acid-pressurized liquid extraction (PLE) has been developed for the simultaneous extraction of major and trace elements (As, Ca, Cd, Co, Cr, K, Mg, Mn, Na, Pb, Sr and Zn) from edible Seaweeds. The target elements have been simultaneously determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The influence of several extraction parameters (e.g. acetic acid concentration, extraction temperature, extraction time, pressure, number of cycles, particle size and diatomaceous earth (DE) mass/sample mass ratio) on the efficiency of metal leaching has been evaluated. The results showed that metal extraction efficiency depends on the mass ratio of the dispersing agent mass and the sample. The optimized procedure consisted of the following conditions: acetic acid (0.75 M) as an extracting solution, 5 min of extraction time, one extraction cycle at room temperature at a pressure of 10.3 MPa and addition of a dispersing agent (at a ratio of 5:1 over the sample mass). The leaching procedure was completed after 7 min (5 min extraction time plus 1 min purge time plus 1 min end relief time). Limits of detection and quantification and repeatability of the over all procedure have been assessed. Method validation was performed analysing two Seaweed reference materials (NIES-03 Chlorella Kessleri and NIES-09 Sargasso). The developed extraction method has been applied to red (Dulse and Nori), green (Sea Lettuce) and brown (Kombu, Wakame and Sea Spaghetti) edible Seaweeds.
Keizo Yuasa - One of the best experts on this subject based on the ideXlab platform.
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comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the Sea hare aplysia kurodai unique properties of two α amylases and two α glucosidases
FEBS Open Bio, 2014Co-Authors: Akihiko Tsuji, Nami Nishiyama, Miki Ohshima, Saori Maniwa, Shuji Kuwamura, Masataka Shiraishi, Keizo YuasaAbstract:Sea Lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the Sea hare Aplysia kurodai, shows a clear feeding preference for Sea Lettuce. Compared with cellulose, Sea Lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on Sea Lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When Sea Lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, Sea hares efficiently digest Sea Lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes.
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comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea hare aplysia kurodai efficient glucose release from Sea Lettuce by synergistic action of 45 kda endoglucanase and 210 kda s glucosidase
PLOS ONE, 2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:Although many endo-s-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on Seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and Seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the Sea hare (Aplysia kurodai) were screened and characterized to determine how the Sea hare obtains glucose from Sea Lettuce (Ulva pertusa). Four endo-s-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 s-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K s-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-s-glucoside, 4MU-s-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K s-glucosidase possesses lactase activity. Although s-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K s-glucosidase. Kinetic analysis of the inhibition of 210K s-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of Sea Lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K s-glucosidase. Our results indicate that 45K cellulase and 210K s-glucosidase are the core components of the Sea hare digestive system for efficient production of glucose from Sea Lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from Seaweed.
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Hydrolysis of CMC, filter paper, and Seaweeds by the synergistic action of cellulases and ß-glucosidases.
2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, Sea Lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of Sea Lettuce treated with purified enzymes or Trichoderma cellulase. Control Sea Lettuce (E) and Sea Lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.
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Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea hare Aplysis kurodai. Efficient glucose release from Sea Lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa b-glucosidase. PLoS One 8
2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on Seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and Seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the Sea hare (Aplysia kurodai) were screened and characterized to determine how the Sea hare obtains glucose from Sea Lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21 K, 45 K, 65 K, and 95 K cellulase) and 2 ß-glucosidases (110 K and 210 K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21 K cellulase, whereas 45 K and 65 K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210 K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210 K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210 K ß-glucosidase. Kinetic analysis of the inhibition of 210 K ß-glucosidase b
Cristina Garciasartal - One of the best experts on this subject based on the ideXlab platform.
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study of cooking on the bioavailability of as co cr cu fe ni se and zn from edible Seaweed
Microchemical Journal, 2013Co-Authors: Cristina Garciasartal, Antonio Moredapineiro, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract This study examines the effects of cooking on the bioavailability of As, Co, Cr, Cu, Fe, Ni, Se and Zn from edible Seaweed. Four edible Galician Seaweeds (Kombu, Wakame, Sea Lettuce and Nori) were cooked following the manufacturer's recommendations. Cr, Fe, Co, Ni, Cu, Zn, As and Se were analyzed in raw and cooked Seaweeds after subjecting them to an acid digestion. The metals were released into the cooking water during the heat treatment with the exception of Ni and Zn in Kombu, Cr, Fe and Co in Wakame, and Zn in Nori, for which almost 100% was retained in the Seaweed. An in vitro digestion method was developed to assess the bioavailability percentages of those metals in the cooked Seaweed. Results suggested that Fe, Zn and Cu are not bioavailable for body functions in the studied matrices. Low dialyzability percentages were found for Cr, Co, Ni, As and Se, ranging from 1.0–16.0%, 3.4–27%, 8.2–12.9%, 9.7–21.0%, and 14.6–61.3%, respectively. Kombu Seaweed was used as a model to perform a mass balance study for evaluating the accuracy of the bioavailability study of cooked Seaweed. Standard deviation homogeneity was corroborated by means of the Cochran's and Bartlett's statistical tests (p-values > 0.05). An ANOVA test was used to compare the mean metal concentrations between and within groups. p-Values higher than 0.05 showed that there were no statistical differences, and the mass balance was adequate. Finally, the influence of the main food constituents (fat, protein, dietary fiber and carbohydrate) on the metal bioavailability was evaluated. Correlation matrix and multivariate analysis based on multiple linear regression indicated positive and negative correlations between the type of Seaweed and metal concentration.
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two dimensional hplc coupled to icp ms and electrospray ionisation esi ms ms for investigating the bioavailability in vitro of arsenic species from edible Seaweed
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Cristina Garciasartal, Pilar Bermejobarrera, Sutthinun Taebunpakul, Emma Stokes, M C Barcielaalonso, Heidi GoenagainfanteAbstract:Edible Seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible Seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible Seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four Seaweed namely kombu, wakame, nori and Sea Lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1 h sonication in a water bath followed by HPLC-ICP-MS resulted in 40-61% of the total As to be found in the buffered aqueous extract, of which 86-110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was Sea Lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in Sea Lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11-16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93-120% and 41% of the dialyzable As fraction for kombu, wakame, nori and Sea Lettuce, respectively. Moreover, the relative As species distribution in Seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in Seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in Sea Lettuce samples was also confirmed.
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use of an in vitro digestion method to evaluate the bioaccessibility of arsenic in edible Seaweed by inductively coupled plasma mass spectrometry
Microchemical Journal, 2011Co-Authors: Cristina Garciasartal, Raquel Dominguezgonzalez, Antonio Moredapineiro, M C Barcielaalonso, Vanessa Romarishortas, Pilar BermejobarreraAbstract:Abstract Different types of edible Seaweeds (Kombu, Wakame, Nori and Sea Lettuce) harvested in the Galician coast (Northwestern Spain) were analyzed for total arsenic quantification before and after being cooked following manufacturer's instructions. Furthermore, the total arsenic in the bioaccessible fraction obtained after simulating a human digestion by an in vitro process was determined in those raw and cooked Seaweeds. The detection of the target element was performed by inductively coupled plasma-mass spectrometry (ICP-MS) which was equipped with a collision cell to avoid polyatomic interferences. Piperazine-NN-bis (2-ethane-sulfonic acid) disodium (PIPES) buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut-off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a BCR-279 certified reference material. The accuracy of the in vitro procedures was established by a mass balance study for Nori and Sea Lettuce which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). Results showed that the effect of cooking Seaweed causes the removal of the element into the cooking water. Dialyzability percentages found in raw Seaweed samples were comparable to those found in cooked Seaweed, except in the case of Sea Lettuce sample for which a lower dialyzability percentage was found when it was cooked.
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bioavailability study using an in vitro method of iodine and bromine in edible Seaweed
Food Chemistry, 2011Co-Authors: Vanessa Romaris Hortas, Raquel Dominguezgonzalez, Antonio Moredapineiro, Cristina Garciasartal, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract Raw edible Seaweed harvested in the Galician coast (Northwester Spain), including two red Seaweed types (Dulse and Nori), three brown Seaweed (Kombu, Wakame and Sea Spaghetti), one green Seaweed (Sea Lettuce) and one microalgae ( Spirulina platensis ) were analysed for total iodine and total bromine, as well as for iodine and bromine bioavailability by in-vitro methods (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked Seaweed sample (canned in brine) consisting of a mixture of two brown Seaweed (Sea Spaghetti and Furbelows) and a derived product (agar–agar) from the red Seaweed Gelidiumm sesquipedale , were also included in the study. All measurements were carried out by inductively coupled plasma–mass spectrometry using tellurium and yttrium as internal standards for iodine and bromine, respectively. An optimised microwave assisted alkaline (TMAH) digestion procedure was used as sample pre-treatment for total iodine and bromine determinations, as well as for the determination of both elements in the non-dialyzable fractions. PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for the intestinal digestion. Accuracy of the method (total bromine and iodine determinations) was assessed by analysing a NIES-09 certified reference material. The accuracy of the in-vitro procedure was established by a mass-balance study which led, after statistical evaluation (95% confidence interval), good accuracy of the whole in-vitro process. The highest dialyzability bromine percentages (36 ± 0.7% and 47 ± 3.0%) were obtained for red Seaweed (Dulse and Nori), while higher dialyzability iodine was assessed for the brown Seaweed (Kombu), around 17% ± 0.7%.
Akihiko Tsuji - One of the best experts on this subject based on the ideXlab platform.
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comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the Sea hare aplysia kurodai unique properties of two α amylases and two α glucosidases
FEBS Open Bio, 2014Co-Authors: Akihiko Tsuji, Nami Nishiyama, Miki Ohshima, Saori Maniwa, Shuji Kuwamura, Masataka Shiraishi, Keizo YuasaAbstract:Sea Lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the Sea hare Aplysia kurodai, shows a clear feeding preference for Sea Lettuce. Compared with cellulose, Sea Lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on Sea Lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When Sea Lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, Sea hares efficiently digest Sea Lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes.
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comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea hare aplysia kurodai efficient glucose release from Sea Lettuce by synergistic action of 45 kda endoglucanase and 210 kda s glucosidase
PLOS ONE, 2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:Although many endo-s-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on Seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and Seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the Sea hare (Aplysia kurodai) were screened and characterized to determine how the Sea hare obtains glucose from Sea Lettuce (Ulva pertusa). Four endo-s-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 s-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K s-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-s-glucoside, 4MU-s-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K s-glucosidase possesses lactase activity. Although s-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K s-glucosidase. Kinetic analysis of the inhibition of 210K s-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of Sea Lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K s-glucosidase. Our results indicate that 45K cellulase and 210K s-glucosidase are the core components of the Sea hare digestive system for efficient production of glucose from Sea Lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from Seaweed.
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Hydrolysis of CMC, filter paper, and Seaweeds by the synergistic action of cellulases and ß-glucosidases.
2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, Sea Lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of Sea Lettuce treated with purified enzymes or Trichoderma cellulase. Control Sea Lettuce (E) and Sea Lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.
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Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea hare Aplysis kurodai. Efficient glucose release from Sea Lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa b-glucosidase. PLoS One 8
2013Co-Authors: Akihiko Tsuji, Nami Nishiyama, Keiko Tominaga, Keizo YuasaAbstract:Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on Seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and Seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the Sea hare (Aplysia kurodai) were screened and characterized to determine how the Sea hare obtains glucose from Sea Lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21 K, 45 K, 65 K, and 95 K cellulase) and 2 ß-glucosidases (110 K and 210 K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21 K cellulase, whereas 45 K and 65 K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210 K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU–ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210 K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210 K ß-glucosidase. Kinetic analysis of the inhibition of 210 K ß-glucosidase b
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study of cooking on the bioavailability of as co cr cu fe ni se and zn from edible Seaweed
Microchemical Journal, 2013Co-Authors: Cristina Garciasartal, Antonio Moredapineiro, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract This study examines the effects of cooking on the bioavailability of As, Co, Cr, Cu, Fe, Ni, Se and Zn from edible Seaweed. Four edible Galician Seaweeds (Kombu, Wakame, Sea Lettuce and Nori) were cooked following the manufacturer's recommendations. Cr, Fe, Co, Ni, Cu, Zn, As and Se were analyzed in raw and cooked Seaweeds after subjecting them to an acid digestion. The metals were released into the cooking water during the heat treatment with the exception of Ni and Zn in Kombu, Cr, Fe and Co in Wakame, and Zn in Nori, for which almost 100% was retained in the Seaweed. An in vitro digestion method was developed to assess the bioavailability percentages of those metals in the cooked Seaweed. Results suggested that Fe, Zn and Cu are not bioavailable for body functions in the studied matrices. Low dialyzability percentages were found for Cr, Co, Ni, As and Se, ranging from 1.0–16.0%, 3.4–27%, 8.2–12.9%, 9.7–21.0%, and 14.6–61.3%, respectively. Kombu Seaweed was used as a model to perform a mass balance study for evaluating the accuracy of the bioavailability study of cooked Seaweed. Standard deviation homogeneity was corroborated by means of the Cochran's and Bartlett's statistical tests (p-values > 0.05). An ANOVA test was used to compare the mean metal concentrations between and within groups. p-Values higher than 0.05 showed that there were no statistical differences, and the mass balance was adequate. Finally, the influence of the main food constituents (fat, protein, dietary fiber and carbohydrate) on the metal bioavailability was evaluated. Correlation matrix and multivariate analysis based on multiple linear regression indicated positive and negative correlations between the type of Seaweed and metal concentration.
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two dimensional hplc coupled to icp ms and electrospray ionisation esi ms ms for investigating the bioavailability in vitro of arsenic species from edible Seaweed
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Cristina Garciasartal, Pilar Bermejobarrera, Sutthinun Taebunpakul, Emma Stokes, M C Barcielaalonso, Heidi GoenagainfanteAbstract:Edible Seaweed consumption is a route of exposure to arsenic. However, little attention has been paid to estimate the bioaccessibility and/or bioavailability of arsenosugars in edible Seaweed and their possible degradation products during gastrointestinal digestion. This work presents first use of combined inductively coupled plasma mass spectroscopy (ICP-MS) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) with two-dimensional HPLC (size exclusion followed by anion exchange) to compare the qualitative and quantitative arsenosugars speciation of different edible Seaweed with that of their bioavailable fraction as obtained using an in vitro gastrointestinal digestion procedure. Optimal extraction conditions for As species from four Seaweed namely kombu, wakame, nori and Sea Lettuce were selected as a compromise between As extraction efficiency and preservation of compound identity. For most investigated samples, the use of ammonium acetate buffer as extractant and 1 h sonication in a water bath followed by HPLC-ICP-MS resulted in 40-61% of the total As to be found in the buffered aqueous extract, of which 86-110% was present as arsenosugars (glycerol sugar, phosphate sugar and sulfonate sugar for wakame and kombu and glycerol sugar and phosphate sugar for nori). The exception was Sea Lettuce, for which the arsenosugar fraction (glycerol sugar, phosphate sugar) only comprised 44% of the total extracted As. Interestingly, the ratio of arsenobetaine and dimethylarsinic acid to arsenosugars in Sea Lettuce extracts seemed higher than that for the rest of investigated samples. After in vitro gastrointestinal digestion, approximately 11-16% of the total As in the solid sample was found in the dialyzates with arsenosugars comprising 93-120% and 41% of the dialyzable As fraction for kombu, wakame, nori and Sea Lettuce, respectively. Moreover, the relative As species distribution in Seaweed-buffered extracts and dialyzates was found to be very similar. Collection of specific fractions from the size-exclusion column to be analysed using anion-exchange HPLC-ESI-MS/MS enabled improved chromatographic selectivity, particularly for the less retained arsenosugar (glycerol sugar), facilitating confirmation of the presence of arsenosugars in Seaweed extracts and dialyzates. Using this approach, the presence of arsenobetaine in Sea Lettuce samples was also confirmed.
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use of an in vitro digestion method to evaluate the bioaccessibility of arsenic in edible Seaweed by inductively coupled plasma mass spectrometry
Microchemical Journal, 2011Co-Authors: Cristina Garciasartal, Raquel Dominguezgonzalez, Antonio Moredapineiro, M C Barcielaalonso, Vanessa Romarishortas, Pilar BermejobarreraAbstract:Abstract Different types of edible Seaweeds (Kombu, Wakame, Nori and Sea Lettuce) harvested in the Galician coast (Northwestern Spain) were analyzed for total arsenic quantification before and after being cooked following manufacturer's instructions. Furthermore, the total arsenic in the bioaccessible fraction obtained after simulating a human digestion by an in vitro process was determined in those raw and cooked Seaweeds. The detection of the target element was performed by inductively coupled plasma-mass spectrometry (ICP-MS) which was equipped with a collision cell to avoid polyatomic interferences. Piperazine-NN-bis (2-ethane-sulfonic acid) disodium (PIPES) buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut-off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a BCR-279 certified reference material. The accuracy of the in vitro procedures was established by a mass balance study for Nori and Sea Lettuce which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). Results showed that the effect of cooking Seaweed causes the removal of the element into the cooking water. Dialyzability percentages found in raw Seaweed samples were comparable to those found in cooked Seaweed, except in the case of Sea Lettuce sample for which a lower dialyzability percentage was found when it was cooked.
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bioavailability study using an in vitro method of iodine and bromine in edible Seaweed
Food Chemistry, 2011Co-Authors: Vanessa Romaris Hortas, Raquel Dominguezgonzalez, Antonio Moredapineiro, Cristina Garciasartal, M C Barcielaalonso, Pilar BermejobarreraAbstract:Abstract Raw edible Seaweed harvested in the Galician coast (Northwester Spain), including two red Seaweed types (Dulse and Nori), three brown Seaweed (Kombu, Wakame and Sea Spaghetti), one green Seaweed (Sea Lettuce) and one microalgae ( Spirulina platensis ) were analysed for total iodine and total bromine, as well as for iodine and bromine bioavailability by in-vitro methods (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked Seaweed sample (canned in brine) consisting of a mixture of two brown Seaweed (Sea Spaghetti and Furbelows) and a derived product (agar–agar) from the red Seaweed Gelidiumm sesquipedale , were also included in the study. All measurements were carried out by inductively coupled plasma–mass spectrometry using tellurium and yttrium as internal standards for iodine and bromine, respectively. An optimised microwave assisted alkaline (TMAH) digestion procedure was used as sample pre-treatment for total iodine and bromine determinations, as well as for the determination of both elements in the non-dialyzable fractions. PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for the intestinal digestion. Accuracy of the method (total bromine and iodine determinations) was assessed by analysing a NIES-09 certified reference material. The accuracy of the in-vitro procedure was established by a mass-balance study which led, after statistical evaluation (95% confidence interval), good accuracy of the whole in-vitro process. The highest dialyzability bromine percentages (36 ± 0.7% and 47 ± 3.0%) were obtained for red Seaweed (Dulse and Nori), while higher dialyzability iodine was assessed for the brown Seaweed (Kombu), around 17% ± 0.7%.