ß-Glucan fragmentation by microfluidization and TNF-α-immunostimulating activity of fragmented ß-glucans.

Fragmentation of ß-glucans secreted by the fungus Ophiocordyceps dipterigena BCC 2073 achieved by microfluidization was investigated. The degree of ß-glucan fragmentation was evaluated based on the average number of chain scissions (α). The effects on the α value of experimental variables like solid concentration of the ß-glucan suspension, interaction chamber pressure, and number of passes through the microfluidizer were examined. Kinetic studies were conducted using the relationships of the α and suspension viscosity values with the number of passes. Evidence indicated that α increases with the interaction chamber pressure and the number of passes, whereas the solid concentration shows the inverted effect. Kinetic data indicated that the fragmentation rate increases with ß-glucan solid concentration and interaction chamber pressure. Furthermore, since ß-glucan molecular weight is a key factor determining its biological activity, the effect of ß-glucans of different molecular weights produced by fragmentation on tumor necrosis factor (TNF)-α-stimulating activity in THP-1 human macrophage cells was investigated. Evidence suggested that ß-glucans have an immunostimulating effect on macrophage function, in the absence of cytotoxic effects. Indeed, ß-glucans characterized by a range of molecular weights produced via microfluidization exhibited promise as immunostimulatory agents.

Vegetative insecticidal protein (Vip3A) production by Bacillus thuringiensis Bt294 and its efficacy against Lepidopteran pests (Spodoptera exigua).

A vegetative insecticidal protein, Vip3A, is highly active against lepidopteran pests, which are the most important pests in most tropical countries. An important aspect of the successful commercial production of this bacterial insecticide is the development of bacterial culture media that maximize the titres of this protein and cost reduction. This study aimed to investigate and optimize Vip3A production by Bacillus thuringiensis Bt294 using statistical methods and 3-step sequential approaches. The experimental design showed that the production of Vip3A was maximized to 300 mg/L when the bacterium was cultivated in medium composed of 5.05 g/L glycerol, 49.17 g/L soytone, 30.05 g/L casein hydrolysate, 1.99 g/L CaCl2.2H2O, 7.5 mg/L CuSO4, 15 mg/L MnSO4.H2O, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 0.2 g/L MgSO4.7H2O, 5 g/L yeast extract, 2.5 mg/L NiCl2.6H2O and 3 mL/L vitamin solution. B. thuringiensis Bt294 Vip3A toxin was highly toxic to Spodoptera exigua with LC50 values of 187.1 ng/cm2 at 7 days. This result demonstrated that a high titre of Vip3A produced by B. thuringiensis Bt294 will be useful as a biological control agent. This optimization will allow production to be scaled up for commercial production in the future.

Effects of dietary supplementation with different levels and molecular weights of fungal ß-glucan on performances, health and meat quality in broilers

Objective: To investigate the effects of dietary supplementation with different levels and molecular weights of fungal ß-glucan on productive performances, health, carcass traits and meat quality in broilers. Methods: Two hundred and ten of one-day-old chicks with equal sex were assigned to seven experimental groups in 2×4 factorial arrangement. These groups were supplemented with (0, 10, 30 and 60 ppm) of molecular weight 1-3, 1-6 ß-glucan (low or high). High molecular weight ß-glucan (H: 943 kDa) was obtained from Ophiocordyceps dipterigena BCC 2073, whereas H with -Irradiation treatment was performed to achieve low molecular weight ß-glucan (L: 8 kDa). Results: There was no statistical significance in productive performances, apparent digestibility and interaction between fixed factors along 42 days of experiment (P>0.05). A higher caecal amylase activity was present in the group that received L, while there was a dramatic decrease in H and the control groups, respectively (P<0.05). The increase of supplemental dose increased caecal amylase activity (P<0.05). Immunomodulatory effects from L was revealed by the marked increase of phagocytic activity, relative weight of thymus and bursa of fabricius (P<0.05). Similarly, the additive dose at 30 ppm provided the same results, whereas the only significant difference with supplementation at 60 ppm was an increase in phagocytic activity (P<0.05). Interestingly, villi height of broilers fed L was higher than other groups (P<0.05). The treatments did not influence haematology, blood chemistry, antibody production level against vaccination, carcass traits and meat quality (P>0.05). Conclusion: The supplementation of L at 30 ppm was suggested to achieve benefits of immune modulation without adverse effects on other parameters.

Pimarane diterpenes from the endophytic fungus Eutypella sp. BCC 13199.

Two new pimarane-type diterpenes, eutypellones A (1) and B (2), were isolated from the endophytic fungus Eutypella sp. BCC 13199. Cytotoxic activities of the pimaranes 1-5, isolated from this fungus, were evaluated.

Exobiopolymer production of Ophiocordyceps dipterigena BCC 2073: optimization, production in bioreactor and characterization.

BACKGROUND: Biopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus Ophiocordyceps dipterigena BCC 2073 produces an exobiopolymer, a (1-->3)-beta-D-glucan, in low quantity under screening conditions. Optimization of O. dipterigena BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study. RESULTS: In order to improve and certify the productivity of this strain, a sequential approach of 4 steps was followed. The first step was the qualitative selection of the most appropriate carbon and nitrogen sources (general factorial design) and the second step was quantitative optimization of 5 physiological factors (fractional factorial design). The best carbon and nitrogen source was glucose and malt extract respectively. From an initial production of 2.53 g x L(-1), over 13 g x L(-1) could be obtained in flasks under the improved conditions (5-fold increase). The third step was cultivation in a 5 L bioreactor, which produced a specific growth rate, biomass yield, exobiopolymer yield and exobiopolymer production rate of 0.014 h-1, 0.32 g x g(-1) glucose, 2.95 g x g biomass(-1) (1.31 g x g(-1) sugar), and 0.65 g x (L x d)(-1), respectively. A maximum yield of 41.2 g x L(-1) was obtained after 377 h, a dramatic improvement in comparison to the initial production. In the last step, the basic characteristics of the biopolymer were determined. The molecular weight of the polymer was in the range of 6.3 x 10(5) - 7.7 x 10(5) Da. The exobiopolymer, at 50 and 100. microg x mL(-1), induced synthesis in normal dermal human fibroblasts of 2227 and 3363 pg x mL(-1) interleukin-8 respectively. CONCLUSIONS: High exobiopolymer yield produced by O. dipterigena BCC 2073 after optimization by qualitative and quantitative methods is attractive for various applications. It induced high IL-8 production by normal dermal fibroblasts, which makes it promising for application as wound healing material. However, there are still other possible applications for this biopolymer, such as an alternative source of biopolymer substitute for hyaluronic acid, which is costly, as a thickening agent in the cosmetic industry due to its high viscosity property, as a moisturizer, and in encapsulation.

Hirsutellone F, a dimer of antitubercular alkaloids from the seed fungus Trichoderma species BCC 7579.

[structure: see text] Hirsutellone F (7), a novel alkaloid dimer, was isolated together with known monomers, hirsutellones A (1), B (2), and C (3), from the seed fungus Trichoderma sp. BCC 7579. The structure of 7 was elucidated by spectroscopic analysis. Studies on biomimetic chemistry, using the dimer 7, suggested that compound 8 (17,1'-dehydrohirsutellone B) should be the precursor for all hirsutellones.

Metabolic control analysis of Aspergillus niger L-arabinose catabolism.

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.

Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus.

Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.