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Effects of antioxidants addition on the marine dinoflagellates biotoxin production
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
2.6.072
2.6.073
Kinetic extraction of nordihygroguaiaretic acid from Larrea tridentata using two different techniques: microwave-assisted extraction and solid-state fermentation
Effects of antioxidants addition on the marine dinoflagellates biotoxin production
S. Martins 1,∗ , C. Aguilar 2 , J.A. Teixeira 1
J.J. Gallardo Rodríguez ∗ , A. Sánchez Mirón, F. García Camacho, M.D.C. Cerón García, E.H. Belarbi Haftalaui, E. Molina Grima ˜ University of Almería, La Canada de San Urbano, Spain
1
University of Minho, Saltillo, Mexico 2 Autonomous University of Coahuila, Saltillo, Mexico
Nordihygroguaiaretic acid (NDGA) is a lignan found in the semidesert plant Larrea tridentata (Zygophyllaceae), known as creosote bush, which grows in the Southwestern United States and Northern Mexico. NDGA has several biological activities, such as antioxidant, antiviral, cancer chemopreventive, and antitumorgenic activities with great importance in the pharmaceutical industry. Extraction of phytochemical compounds from plants has been conventionally performed by heat-reflux systems, which are time consuming and require high amounts of solvents. The increased need for ideal extraction methods that allows the recovery of high yields of a bioactive compound from plant material in a shortest processing time with low costs represents an important challenge. The objective of this work was to extract NDGA from L. tridentata leaves by microwave-assisted extraction (MAE), and an alternative environmentally clean technology such as solid-state fermentation (SSF) using two different strains: A. niger GH1 and A. niger PSH. For MAE dried powdered plant was mixed with an appropriate extraction solvent (methanol in water at 50%, w/w). The suspensions were irradiated with microwaves at a power of 800 W in a pre-setting procedure where after each period of 1 min the sample was allowed to cool at room temperature (total time of 4 min). The extraction temperature was 70 ± 2 ◦ C. SSF was performed using 4.7 ml of Czapek-Dox broth with 2 g of L. tridentata as solid support (carbon source), 2 × 107 spores/gL. tridentata , and an incubation temperature of 30 ◦ C. The fermentation process was carried out at 70% humidity for 96 h, sampling every 12 h. NDGA was quantified by HPLC technique. Results showed that MAE had advantages over the conventional extraction technique by reflux (CER) used to recover bioactive compounds from plant material. In MAE the extraction time was significantly reduced (P < 0.05), and significantly higher (P < 0.05) yields of NDGA were obtained compared to CER (1 and 18 min; 3.79 ± 0.65 and 3.42 ± 0.19%, respectively). Regarding SSF the main disadvantage was the extraction time in comparison with the others methods used (48 h). However, SSF using A. niger PSH showed significantly higher (P < 0.05) yields of NDGA (7.85 ± 2.44%) when compared with those obtained by MAE and CER. In conclusion, SFF showed the highest yields of NDGA demanding longer processing time. Further studies are required in order to visualize differences in biological activities of NDGA extracted by the different methods evaluated. doi:10.1016/j.nbt.2009.06.176
S218
www.elsevier.com/locate/nbt
Marine dinoflagellates are potentially important as sources of novel high-value toxins in biomedical, toxicological and chemical research programs. Prices range from several hundred Euros to more than D 30,000 per mg. A lack of sufficient quantities of toxins for afore-mentioned purposes remains a significant limitation. Mainly because, little is known about the difficulty to culture them in bioreactors. The main obstacle is their sensitivity to shear forces. However, low hydrodynamic forces that avoid cell breakage appeared to induce the production and accumulation of reactive oxygen species (ROS) within the cells, causing peroxidation of cellular lipids and ultimately cell damage. Moreover, oxidative stress in dinoflagellates has been shown to be induced by many factors that could be triggered off in bioreactor cultures. Prevention and mitigation are key issues that need to be cleared up in order to obtain stable production of biomass. Antioxidant additions and perfusion cultures were assayed with monocultures of the red-tide dinoflagellate Protoceratium reticulatum (GG1AM). Fed-batch, continuous and perfusion cultures were carried out in a traditional glass-made 2 L bioreactor. Fed-batch, and perfusion cultures were carried out in a pilot-plant glass-made reactor of 20 L. Biomass and toxin production were measured. Temperature, pH and dissolved oxygen (DO) were recorded online. The maximum cell concentration and productivity obtained was one order of magnitude higher than the maximum previously referenced for this species. Cultures were maintained healthy up to five months. In the pilot-plant scale we were able to get similar productivities per unit of volume than in the lab-scale bioreactor. doi:10.1016/j.nbt.2009.06.177
2.6.074 Influence of turbulence on the adaptation of the baculovirus-producer Spodoptera exigua Se301 cell line to suspension culture A. Beas-Catena ∗ , A. Sánchez-Mirón, F. García-Camacho, E. MolinaGrima ˜ University of Almeria, La Canada de San Urbano, Spain
One of the main pests affecting global crops is insects from the order Lepidoptera. In the greenhouses of Almeria (Southern Spain) one of the most important pest is Spodoptera exigua, which feeds mainly on peppers and watermelons causing important damage to crops and economic losses. The way to deal with plagues have been the use of chemical pesticides of broad-spectrum; however, as they are being banned, they must be replaced with other means of control safer to people and to the environment. One of the
ABSTRACTS
2.6.072
2.6.073
Kinetic extraction of nordihygroguaiaretic acid from Larrea tridentata using two different techniques: microwave-assisted extraction and solid-state fermentation
Effects of antioxidants addition on the marine dinoflagellates biotoxin production
S. Martins 1,∗ , C. Aguilar 2 , J.A. Teixeira 1
J.J. Gallardo Rodríguez ∗ , A. Sánchez Mirón, F. García Camacho, M.D.C. Cerón García, E.H. Belarbi Haftalaui, E. Molina Grima ˜ University of Almería, La Canada de San Urbano, Spain
1
University of Minho, Saltillo, Mexico 2 Autonomous University of Coahuila, Saltillo, Mexico
Nordihygroguaiaretic acid (NDGA) is a lignan found in the semidesert plant Larrea tridentata (Zygophyllaceae), known as creosote bush, which grows in the Southwestern United States and Northern Mexico. NDGA has several biological activities, such as antioxidant, antiviral, cancer chemopreventive, and antitumorgenic activities with great importance in the pharmaceutical industry. Extraction of phytochemical compounds from plants has been conventionally performed by heat-reflux systems, which are time consuming and require high amounts of solvents. The increased need for ideal extraction methods that allows the recovery of high yields of a bioactive compound from plant material in a shortest processing time with low costs represents an important challenge. The objective of this work was to extract NDGA from L. tridentata leaves by microwave-assisted extraction (MAE), and an alternative environmentally clean technology such as solid-state fermentation (SSF) using two different strains: A. niger GH1 and A. niger PSH. For MAE dried powdered plant was mixed with an appropriate extraction solvent (methanol in water at 50%, w/w). The suspensions were irradiated with microwaves at a power of 800 W in a pre-setting procedure where after each period of 1 min the sample was allowed to cool at room temperature (total time of 4 min). The extraction temperature was 70 ± 2 ◦ C. SSF was performed using 4.7 ml of Czapek-Dox broth with 2 g of L. tridentata as solid support (carbon source), 2 × 107 spores/gL. tridentata , and an incubation temperature of 30 ◦ C. The fermentation process was carried out at 70% humidity for 96 h, sampling every 12 h. NDGA was quantified by HPLC technique. Results showed that MAE had advantages over the conventional extraction technique by reflux (CER) used to recover bioactive compounds from plant material. In MAE the extraction time was significantly reduced (P < 0.05), and significantly higher (P < 0.05) yields of NDGA were obtained compared to CER (1 and 18 min; 3.79 ± 0.65 and 3.42 ± 0.19%, respectively). Regarding SSF the main disadvantage was the extraction time in comparison with the others methods used (48 h). However, SSF using A. niger PSH showed significantly higher (P < 0.05) yields of NDGA (7.85 ± 2.44%) when compared with those obtained by MAE and CER. In conclusion, SFF showed the highest yields of NDGA demanding longer processing time. Further studies are required in order to visualize differences in biological activities of NDGA extracted by the different methods evaluated. doi:10.1016/j.nbt.2009.06.176
S218
www.elsevier.com/locate/nbt
Marine dinoflagellates are potentially important as sources of novel high-value toxins in biomedical, toxicological and chemical research programs. Prices range from several hundred Euros to more than D 30,000 per mg. A lack of sufficient quantities of toxins for afore-mentioned purposes remains a significant limitation. Mainly because, little is known about the difficulty to culture them in bioreactors. The main obstacle is their sensitivity to shear forces. However, low hydrodynamic forces that avoid cell breakage appeared to induce the production and accumulation of reactive oxygen species (ROS) within the cells, causing peroxidation of cellular lipids and ultimately cell damage. Moreover, oxidative stress in dinoflagellates has been shown to be induced by many factors that could be triggered off in bioreactor cultures. Prevention and mitigation are key issues that need to be cleared up in order to obtain stable production of biomass. Antioxidant additions and perfusion cultures were assayed with monocultures of the red-tide dinoflagellate Protoceratium reticulatum (GG1AM). Fed-batch, continuous and perfusion cultures were carried out in a traditional glass-made 2 L bioreactor. Fed-batch, and perfusion cultures were carried out in a pilot-plant glass-made reactor of 20 L. Biomass and toxin production were measured. Temperature, pH and dissolved oxygen (DO) were recorded online. The maximum cell concentration and productivity obtained was one order of magnitude higher than the maximum previously referenced for this species. Cultures were maintained healthy up to five months. In the pilot-plant scale we were able to get similar productivities per unit of volume than in the lab-scale bioreactor. doi:10.1016/j.nbt.2009.06.177
2.6.074 Influence of turbulence on the adaptation of the baculovirus-producer Spodoptera exigua Se301 cell line to suspension culture A. Beas-Catena ∗ , A. Sánchez-Mirón, F. García-Camacho, E. MolinaGrima ˜ University of Almeria, La Canada de San Urbano, Spain
One of the main pests affecting global crops is insects from the order Lepidoptera. In the greenhouses of Almeria (Southern Spain) one of the most important pest is Spodoptera exigua, which feeds mainly on peppers and watermelons causing important damage to crops and economic losses. The way to deal with plagues have been the use of chemical pesticides of broad-spectrum; however, as they are being banned, they must be replaced with other means of control safer to people and to the environment. One of the