Fatty acid profiling in the genus Pinus in relation to its chemotaxonomy and nutritional or pharmaceutical properties.

To develop and utilize the oil of Pinus seeds and explore natural resources rich in pinolenic acid (PNLA), twenty-one Pinus taxa were evaluated in a search of Δ5-unsaturated polymethylene-interrupted fatty acids (Δ5-UPIFA)-rich oils. While the fatty acid (FA) composition was determined by GC-FID and GC-MS, NMR of crude oils proved to be a fast method for establishing the ratio between Δ5-UPIFA and total FA. For all analyzed taxa, both the geographical origin and the concentration of total FA in the seeds are provided. PNLA and sciadonic acids occurred in all samples, while taxoleic and bishomopinolenic acids were present in most taxa. PNLA reached a maximum of 28.3% of total FA in P. mugo, and P. koraiensis showed the highest total FA amount (66.8 g/100 g seeds). The previously unanalyzed taxon P. ponderosa var. scopulorum can be considered a new PNLA source (17.1%). Principal Component Analysis showed that the similarities in FA profiles allow the grouping of the various taxa within Pinus subsections and confirmed the differential metabolic activities of Δ5 and Δ9 desaturase enzymes. This study showed that several underutilized Pinus taxa could be developed into renewable woody oil species, and their seeds could be used as raw materials for Δ5-UPIFA-rich oils extraction.

Buglossoides spp. seeds, a land source of health-promoting n-3 PUFA and phenolic compounds.

Ahiflower oil© is extracted from the seeds of Buglossoides arvensis, which contains high amounts of stearidonic acid (SDA, 18:4n-3), while its phenolic composition still is unreported. Moreover, several Buglossoides taxa remain unstudied and could become natural sources of SDA. In this work, seeds of several Buglossoides taxa and Ahiflower oil© were screened for fatty acids, phenolic compounds, and in vitro antiproliferative activities against colorectal cancer cells. Four flavonoids and 16 phenolic acids were identified and quantified. Among Buglossoides taxa, the highest amounts of phenolic compounds were found in samples collected in Spain, under a warm Mediterranean climate. Rosmarinic and lithospermic acids were the main phenols found in Buglossoides seeds. The MTT assay showed dose- and time-dependent inhibitory effects of B. arvensis extracts on HT-29 cancer cells, with a GI50value of ∼280 µg/mL after 72 h of cell exposure to seed extracts. The latter showed lower antiproliferative activity than that of pure phenolics due to the simultaneous presence of other compounds in the extracts, as evidenced by 1H NMR. This work constitutes the first approach to evaluate the seeds of several Buglossoides taxa as functional oils-providers to use them as functional foods.

Gamma-linolenic Acid from Fifty-seven Ribes Species and Cultivars.

γ-linolenic acid (GLA, 18:3n-6) is a bioactive fatty acid (FA) that exerts several healthy actions; however, its occurrence is restricted to a few oils. The goal of this study was to detect GLA-rich Ribes species and cultivars (cv), and to achieve this the seeds of 7 Ribes taxa and 50 Ribes cv were surveyed for FA profiles. The highest GLA percentages were found in R. nigrum cv 'Plotnokistnaya', 'Volshebnica', 'Atlant' and 'Nara' (22.6, 22.1, 20.9, and 20.0% of total FA, respectively) and also in R. komarovii (19.6%) and R. nigrum var. sibiricum (18.3%). Stearidonic acid (SDA, 18:4n-3) had the highest values in both R. rubrum 'Konstantinovskaya' and R. niveum 'Smolyaninovskaya' (4.8%). GLA content ranged from 0.4 in some R. rubrum cv and R. niveum 'Smolyaninovskaya' to 3.5 g/100 g seeds in R. nigrum 'Plotnokistnaya'. Principal component analysis (PCA) was performed using PUFA profiles, which allowed grouping Ribes sections as well as black currant cv derived from different pedigree within the section Coreosma. All taxa and cv checked here are valuable by-product sources, given the high GLA percentages contained in their seed oils. Such cv could be used for healthy oils production, as well as for breeding to obtain new cv with improved GLA concentrations.

Mertensia (Boraginaceae) seeds are new sources of γ-linolenic acid and minor functional compounds.

In this work, seeds from selected Mertensia species were analyzed for γ-linolenic acid-rich oils and minor functional compounds. Fatty acids (FA) were analyzed by GC-FID; tocopherols, sterols, squalene, and phenolics compounds by HPLC-DAD, and the structures of the latter were confirmed by LC-MS. M. maritima spp. asiatica and M. sibirica showed the highest amounts of γ-linolenic acid (22.8 and 18.7%, respectively) and total FA (15.9 and 10.9 g/100 g seeds, respectively). M. sibirica had the greatest levels of sterols, phenolics and tocopherols (244.8, 243.9 and 66.3 mg/100 g, respectively), in which stigmasterol, rosmarinic acid and α-tocopherol (237.7, 180.1 and 53.6 mg/100 g, respectively) were the most abundant components. M. maritima spp. asiatica and M. arizonica showed the highest amounts of squalene (2.5 and 1.1 mg /100 g seeds). Mertensia species constitute a new source of GLA-rich oils, suitable to be marketed by the pharmaceutical and food industries.

Simultaneous effect of temperature and irradiance on growth and okadaic acid production from the marine dinoflagellate Prorocentrum belizeanum.

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I0). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·(m-2)·s(-1)), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ(m)) was 0.204 day(-1) at 25 °C and 40 µE·(m-2)·s(-1). Photo-inhibition was observed at I0 > 40 µEm(-2)s(-1), leading to culture death at 120 µE·m(-2)·s(-1) and 28 °C. Cells at I0 ≥ 80 µE·m(-2)·s(-1) were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ(m)-I0 curves and another empirical model for relating µ(m)-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I0-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.

Induction of CD40 expression and enhancement of monoclonal antibody production on murine B cell hybridomas by cross-linking of IgG receptors.

The 55-6 murine B cell hybridoma line not constitutively expressing CD40 was treated with increasing amounts of intact anti-mouse surface immunoglobulin G antibody (anti-mIgG) either not preincubated or preincubated for 48 h with lipopolysaccharide (LPS). In vitro, cross-linking of surface immunoglobulin G (sIgG) with the whole molecule of anti-IgG antibodies induced the expression of CD69, CD40, and CD19 surface antigens on 55-6 cells. The effect of sIgG ligation was dose-dependent, and preincubation with LPS enhanced their responsiveness to anti-mIgG stimulation. The expression of these surface molecules reached the maximum value during the first part of the cell cycle, corresponding to the position of the G1 peak of the DNA distribution. Stimulation of cells with anti-mIgG did not induce changes either in the number of viable cells or in the fraction of cells undergoing proliferation (mitosis). However, preincubation of 55-6 cells with LPS for 48 h before stimulation with anti-mIgG increased both the maximum specific growth rate (micromax) and the percentage of cells in the G2/M phase, in comparison with non-preincubated cells. Moreover, on cells preincubated with LPS prior to anti-mIgG treatment, specific IgG2a production rate was enhanced significantly compared to that obtained in control cultures. The correlation between the antibody production rate and the amount of IgG that is detectable on the cell surface was analyzed by flow cytometry. A good correlation between secreted and surface IgG was observed, and the results of cell cycle analyses demonstrated that the 55-6 hybridoma cell line has a substantially higher sIgG content in G1 phase.

New culture approaches for yessotoxin production from the dinoflagellate Protoceratium reticulatum.

Fed-batch and perfusion cultures were carried out in a traditional glass 2-L bioreactor with the toxic dinoflagellate Protoceratium reticulatum. The maximum cell concentration obtained was 2.3 x 105 cell.mL-1, which is almost 1 order of magnitude higher than the maximum previously referenced for this species. L1 medium was shown to be clearly deficient in nitrate and phosphate for this strain, and addition of highly concentrated aliquots of these nutrients allowed higher cell concentrations to be obtained. This species consumed high amounts of nitrate and phosphate, 2.1 x 10-3 and 2.3 x 10-4 micromol.h-1.cell-1, respectively. However, this consumption produced a very low number of cells compared to other classes of microalgae, indicating that this species is, like other dinoflagellates, a poor competitor in terms of utilization of inorganic nutrients. Higher production of toxins and pigments was strongly associated with cell number in the culture, with maximum values of 700 ng.mL-1 and 1321 microg.mL-1, respectively. Most yessotoxins remained within the cells and not in the cell-free culture medium, and their production was not related to either the age of the culture or the cell growth phase.

Pilot-plant-scale outdoor mixotrophic cultures of Phaeodactylum tricornutum using glycerol in vertical bubble column and airlift photobioreactors: studies in fed-batch mode.

Pilot-scale (0.19 m column diameter, 2 m tall, and 60 L working volume) outdoor vertical bubble column (BC) and airlift photobioreactors (a split-cylinder (SC) and a draft-tube airlift device (DT)) were compared for fed-batch mixotrophic culture of the microalga Phaeodactylum tricornutum UTEX 640. The cultures were started photoautotrophically until the onset of a quasi-steady-state biomass concentration of 3.4 g L(-)(1). After this, the cultures were supplemented with organic nutrient (glycerol 0.1 M) and a reduced nitrogen source, resulting in an immediate growth rate boost, which was repeated with successive additions of nutrients in all three photobioreactors. During this period the biomass productivity was enhanced compared to photoautotrophic cultures in the three reactors, although differences were found among them. These could be attributed to the different hydrodynamic behavior influencing the transport phenomena inside the cultures. A 25.4 g L(-)(1) maximum biomass concentration was attained in the SC. Further additions of nutrients did not promote any more growth. The consumption of glycerol was quantitative in the first additions but slowed at high biomass concentration, suggesting that a minimum amount of light is needed to sustain growth. No significant effect of the supplied organic nutrient on carotenoids and chlorophylls content was observed, although it had a profound effect on the fatty acid composition. Eicosapentaenoic acid (EPA) content was increased up to 3% (DW) in mixotrophic growth, giving a productivity of 56 mg L(-)(1) d(-)(1), a significant increase compared to the photoautotrophic control, which yielded a maximum EPA content of 1.9% (DW) and a productivity of 18 mg L(-)(1) d(-)(1). The maximum biomass and EPA volumetric yields obtained in this work are comparable with those reported for commercial photoautotrophic monoculture of large quantities of P. tricornutum in closed continuous-run tubular loop bioreactors with tubes that are typically less than 0.08 m in diameter. When the comparison is established in terms of productivities based on the land area occupied, the vertical airlift and bubble-column bioreactors are extraordinarily more productive.

Producing drugs from marine sponges.

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. This review focuses on methods of producing sponge biomass to overcome supply limitations. Production techniques discussed include aquaculture in the sea, the controlled environments of aquariums, and culture of sponge cells and primmorphs. Cultivation in the sea and aquariums are currently the only practicable and relatively inexpensive methods of producing significant quantities of sponge biomass. In the future, metabolite production from cultured sponge cells and primmorphs may become feasible. Obtaining a consistent biomass yield in aquariums requires attention to many factors that are discussed in this work.

Cultivation of explants of the marine sponge Crambe crambe in closed systems.

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.

Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor.

Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum. The food was supplied either as intact algae or as a filtered crude extract. Growth (measured as an increase in underwater weight) was found in both experiments. The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days. The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days. These results show that it is possible to grow a sponge using a single microorganism species as a food source. In addition, it was demonstrated that sponges are also capable of growing on non-particulate food. Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures.