Current Biotechnology - Volume 7, Issue 1, 2018

Background: This is the first report of hyaluronan lyase discovery in filamentous fungi, and the second case of hyaluronan hydrolase production by micromycetes. Hyaluronan (HA) is a glycosaminoglycan composed of Nacetyl- D-glucosamine and D-glucuronic acid. Whereas HA is the major constituent of extracellular matrix and an important regulator of many biological processes in vertebrates, HA degrading enzymes hyaluronidases (HAases) are of particular interest for medicine and pharmacy. Objective: To screen submerged cultures of filamentous fungi for HAase activity, and to characterize the properties of newly discovered HAases. Methods: Fungi were cultivated in bioreactor, enzymes were recovered from the culture broth, and purified by ultrafiltration and chromatography. For enzymes characterization HPLC, LC-ESI-MS, SDS-PAGE, zymography and methods of analytical chemistry were applied. Results: Wood rotting fungus Fistulina hepatica and micromycet Talaromyces stipitatus were identified as producers of extracellular HAases. On the basis of analysis of HA degradation products F. hepatica HAase was identified as HA lyase, while T. stipitatus enzyme was HA hydrolase. The HA lyase had pH optimum close to 4.0 and temperature optimum at around 20°C. T. stipitatus produced acidic HAase with pH optimum at 3.0 and temperature optimum 43°C. T. stipitatus HAase molecular weight was determined, enzyme thermostability, effect of ions on its activity, and substrate specificity were characterized. Conclusion: Filamentous fungi are new and highly promising source of hyaluronidases, which can be used in pharmacy and cosmetics for manufacturing of biologically active HA oligomers.

Background: Cultivation and media optimization is one way to improve hemicellulase production in Aspergillus niger, but bioprocess approaches have limitations due to the carbon catabolite repression (CCR) process. Objective: This study aimed to improve hemicellulase production by genetic modifications at CCR levels in A. niger strain. Method: Deletion of the creA gene to derepress the expression of the hemicellulolytic genes and introduction of a constitutively active version of xlnR were performed. Results and Discussion: The deletion of creA resulted in a higher expression of some hemicellulolytic genes and a higher specific enzyme production. Introduction of the constitutively active xlnR in a ΔCreA strain increased fungal biomass production, but did not result in a higher expression/production of most hemicellulases on xylose, although it was possible to double the productivity of β-xylosidase. Conclusions: A dominant effect of creA deletion on xlnR overexpression in the production of biomass degrading enzymes was present and indicated that a mechanism not totally elucidated is present and involves fungal growth and inductor.

Background: Trichoderma viride is a cellulose-biodegrading fungus which produces high amount of cellulases with a higher ratio of β-glucosidase/total cellulase in its enzyme mixture compared to that in the enzyme complex of T. reesei strains. The culture optimization for cellulase production by T. viride has not been previously studied. Consequently, investigation on the enhancement of its enzyme activity and productivity is a significant aim for research. Methods: A consecutive optimization approach based on statistical experimental designs was applied to improve the production of cellulase enzyme by T. viride through a submerged fermentation. Two-levels Plackett-Burman design (PBD) was employed to screen the significance effects of the different parameters on cellulase production. This was followed by carrying out three levels Box-Behnken design (BBD) to create a polynomial model in order to correlate the relationship between the three tested variables and the cellulase activity. Results: The highest cellulase activity (1.066 IU/mL) was achieved under pH (4), Tween® 80 concentration (0.3 g/L), growth medium volume (30%), CMC concentration (10 g/L), peptone concentration (0.5 g/L), urea concentration (0.2 g/L), harvesting time (4 days), lactose concentration (0.5 g/L) and KH2PO4 concentration (1 g/L). Among the nine variables tested, growth medium volume, peptone concentration and Tween® 80 concentration were selected to be further optimized and modelled via response surface methodology (RSM), due to their significant positive effects on cellulase production. The Maximum predicted and actual cellulase activity (2.8 and 2.9 IU/mL, respectively) was obtained in a 30% growth medium volume containing 1.5 g/L peptone and 0.4 g/L surfactant, with a relatively high correlation between the experimentally observed and predicted values (89%). Conclusion: The most significant experimental factors were observed to be surfactant concentration, growth medium volume and peptone concentration after running the PBD matrix, demonstrating the enhanced level of cellulase activity when any of these factors was increased from its lower to higher level. The revealing of significant variables by PBD and the consequent optimization of their expanded levels by the BBD allowed the enhancement of cellulase activity by 64% (from 1.066 to 2.99 IU/mL).

Background: The focus of this study was the production of a multi-enzyme complex (MC) (xylanase, amylase and cellulase) originating from the fungus Trichoderma harzianum (ALL 42 strain), specific for corn-soybean meal-based substrates, and its subsequent use in the diet of broiler chicks. Methods: Spores of T. harzianum were inoculated in TLE medium containing 0.5% of corn-soybeanbased starter feeds for broiler chicks. After 48 h incubation at 28°C the supernatant was collected and used as source of enzymes. Activities of xylanase, amylase and cellulose were determined and the enzymes were partially characterized. 480 chicks were fed diets supplemented with this MC. These were male, Cobb, with an average starting weight of 50 g, maintained from 1 to 21 days in batteries. The treatments consisted of diets within pre-starter (from 1 to 7 days) and starter (from 8 to 21 days) phases. Food and water availability was unlimited during both experimental periods. Chick weight gain, feed intake, nitrogen intake, nitrogen in excreta, nitrogen balance, nitrogen digestibility and nitrogen retention were determined. Results: The peak in enzyme production was established within 48 hours of culture; the zones of optimal pH were between 2.5-7.5 and optimal temperature was between 40-55°C. The enzymes presented a wide range of thermostability and were highly stable at optimal pH. Significant effects were not observed (P<0.05) regarding performance. There was a significant effect (P>0.05) with higher intake (39.42 g), balance (28.30 g) and retention (45.98) of nitrogen in the pre-starter phase. Conclusion: MC enzymes produced by T. harzianum have potentially application in the development of broiler feeds, since are stable across wide pH and temperature ranges. MC enzymes have not provided performance improvement in the tested birds, but have improved the nitrogen metabolism in the prestarter phase.

Background: In addition to culinary uses, edible and medicinal mushrooms have long been consumed throughout the world as traditional treatments for both acute and chronic human diseases. As a consequence, a great deal of modern research has been conducted with the aim of identifying the compounds responsible for their purported positive health effects. Despite a fairly large body of published data on the potential benefits of fungi-derived dietary supplements (i.e., mushroom extracts, mycelium powders) a consensus on the effects of daily consumption of such supplements on human health has yet to be reached. Although numerous review articles have highlighted the health benefits of edible fungi, few publications provide details about the protocols employed for critically evaluating their composition, nutritional value, quality, metal content, safety, and potential health promoting effects. To help fill this gap, a broad overview of current protocols addressing these areas of research is provided in this review. Methods: We carried out a detailed peer-reviewed article with clearly defined questions, and identified high quality papers that employed standard protocols addressing the key questions we raised. All protocols relevant to nutritional and potential health benefits of edible mushrooms were collected and are summarized here. Results: We identified 219 papers relevant to the review topic. About one fifth of these provided details about a wide variety of fungal secondary metabolites and other metabolites such as polysaccharides and proteins that modulate immune response, leading to positive effects on human health. A few articles covering specific research topics summarized here are: (i) mushrooms and their potential health benefits, (ii) common methods of producing mushrooms and fungal mycelium, (iii) necessity for quality control, (iv) methods used to process mushrooms into various products, (v) protocols for measuring mushroom composition, (vi) antioxidant and total phenolic, flavonoid, and triterpene estimation, (vii) estimation of structural components in mushrooms, (viii) PCR based methods to identify genotype and the presence mycotoxin producing species, and (ix) estimation of microbial load in the mushrooms. Conclusion: The key finding of this review is that targeted chemical analyses, genotyping and other analytical methods will provide important tools to track product quality in terms of both nutritional or medicinal value, and to determine the presence of contaminants. Importantly, this review will also contribute to the understanding of reliable methodologies for assessing product efficacy, composition, quality, and dietary safety of processed mushroom products consumed by the public.

Background: Endophytic fungi isolated from medicinal plants have been recognized as promising sources of highly variable bioactive metabolites. Maytenus ilicifolia, a highly exploited medicinal plant commonly used in popular medicine, is included in the current list of endangered species. Our research group is particularly interested in bioprospecting endophytes from medicinal plants found in Brazil, to control citrus diseases. In this context, Phyllosticta citricarpa is an agronomically important fungus associated with Citrus Black Spot (CBS), a disease associated with large economic losses, mainly due the phytosanitary restrictions for CBS disease in Europe. Objective: The objective of this study was to identify and explore the activity of two endophytic isolates from the medicinal plant M. ilicifolia against P. citricarpa, and to develop a reliable and efficient transformation system for the endophytic strains. Methods: During a screening program on the biodiversity of M. ilicifolia, endophytic strains LGMF1252 and LGMF1253 were isolated and selected for bioprospecting study. The secondary metabolites from LGMF1252 and LGMF1253 were obtained by fermentation in Malt Extract Agar (MEA), and extracted with EtOAC. The antagonistic property was evaluated against mycelial growth and pycnidia formation of the phytopathogen P. citricarpa. A multi-locus sequence analysis using ITS, β- tubulin, and α-actin genes was performed to identify the endophytes LGMF1252 and LGMF1253. Based on these results, the isolate LGMF1252 was transformed via Agrobacterium tumefaciens, with GFP and BAR expression cassettes, using a binary plasmid vector based on pPZP201BK. Results: The secondary metabolites from LGMF1252 and LGMF1253 showed antagonistic properties against mycelial growth and pycnidia formation of P. citricarpa. Multi-locus sequence analysis revealed that the endophytic strains belong to the Xylaria cubensis aggregate. The isolate LGMF1252 was successfully transformed via A. tumefaciens. Conclusion: The inhibition of pycnidia formation can be an alternative for CBS control, considering that there is no effective treatment for the disease and asexual spores play an important role in the development of fruits lesions. Due to the high activity observed, strain LGMF1252 was transformed via A. tumefaciens. The results of this study will facilitate future studies on plant interaction and biological control.

Background: Metarhizium anisopliae controls insect pest populations and commercial formulations are based on conidia, regularly produced in plastic bags using rice grains as substrate. This process is labour- intensive and difficult to control; optimisation represents a current problem, even using different types of bioreactors. However, comparisons between Metarhizium conidia production in bags and packed columns are scarce. Objective: The aim of this study was to evaluate the age of the inoculum and two different type of bioreactors on the production of conidia from Metarhizium anisopliae. Method: Metarhizium anisopliae strain CP-OAX was used for conidia production (inoculum of 19 or 25 days old) using steamed rice as substrate either in plastic bags or packed bed bioreactors, the latter with different sizes with air supply. Results: Up to 30% higher conidia yields were obtained in the packed bed bioreactor after 7 days, using a 25 days old inoculum. Respirometric analysis on the packed bed bioreactor determined lag values from 49.08 to 55.23 h and maximum CO2 production rate of 0.295 ± 0.015 mg gidm-1 h-1; maximum sporulation occurred at 56.7 h. Conclusion: Both inoculum age and bioreactor geometry affect conidia production and productivity. The packed column bioreactor allowed higher conidia production and productivity. Air supply at the high scale packed column bioreactor allows metabolic heat removal to maintain isothermal conditions. In addition, the packed bed bioreactor presents advantages, such as reproducibility, the possibility of scaling-up and monitoring the process in terms of temperature and CO2 production.