Sustainable and Effective Chitosan Production by Dimorphic Fungus Mucor rouxii via Replacing Yeast Extract with Fungal Extract.

The effects of switching morphology and replacing supplementary nutrients with fungal extract (5 and 10 g/L) on the production of major metabolites and chitosan by Mucor rouxii were investigated. This approach was supposed to promote sustainability of the fermentation process and improve its economic feasibility. Different fungal morphologies, i.e., purely filamentous (PF), purely yeast-like (PY), mostly filamentous (MF), and mostly yeast-like (MY), were evaluated. The highest ethanol yields were obtained from the media supplemented with 10 g/L fungal extract for all morphologies, while adding nutrient salts did not make any improvements in these yields, except a slight decrease in the fermentation time. Except for PF morphology, the replacement of yeast extract favored the biomass production yields. Moreover, the alkali insoluble material (AIM) yields were higher as a result of the replacement for most cases. Furthermore, the replacement resulted in increased glucosamine and decreased N-acetyl-glucosamine content of AIM for almost all the morphologies. AIM yields of at least 0.25 g/g-glucose and maximum chitin/chitosan yield of 0.78 g/g-AIM were obtained from the solids remaining after autolysis process, which were higher than that obtained from the raw biomass. The maximum yield of 0.135 g/g-AIM purified chitosan with intact molecular weight was obtained from the biomass with PF morphology supplemented with 10 g/L fungal extract plus nutrients.

Anti-Inflammatory 18ß-Glycyrrhetinin Acid Derivatives Produced by Biocatalysis.

In this study, the biocatalysis of 18ß-glycyrrhetinic acid by two strains of filamentous fungi, namely Rhizopus arrhizus AS 3.2893 and Circinella muscae AS 3.2695, was investigated. Scaled-up biotransformation reactions yielded 14 metabolites. Their structures were established based on extensive nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry data analyses, and seven of them are new compounds. The two fungal strains exhibited distinct biocatalytic features. R. arrhizus could catalyze hydroxylation and carbonylation reactions, whereas C. muscae preferred to catalyze hydroxylation and glycosidation reactions. These highly specific reactions are difficult to achieve by chemical synthesis, particularly under mild conditions. Furthermore, we found that most of the metabolites exhibited pronounced inhibitory activities on lipopolysaccharides-induced nitric oxide production in RAW264.7 cells. These biotransformed derivatives of 18ß-glycyrrhetinic acid could be potential anti-inflammatory agents.

Optimization of conditions for decolorization of azo-based textile dyes by multiple fungal species.

Wastewater from textile industries contains azo dye residues that negatively affect most environmental systems. The biological treatment of these wastes is the best option due to safety and cost concerns. Here we isolated and identified 19 azo dye-degrading fungi and optimized conditions resulting in enhanced degradation. The fungi belonged to five species of Aspergillus and a single Lichtheimia sp. All fungi were evaluated for their ability to decolorize 20 azo dyes. While the most easily transformable azo dye was direct violet (decolorization ranged from 71.1 to 93.3%), the most resistant to decolorization was fast green azo dye. The greatest degradation potential of azo dyes (direct violet and methyl red) was optimized using the most promising four fungal strains and changing media glucose concentration, nitrogen source, and micronutrients. Biomass, lignin peroxidase, and laccases production were also determined in the optimization studies. The decolorization of both azo dyes by the four fungal strains was greatly enhanced by glucose supplementation. The fungal strains were not able to produce lignin peroxidases in the absence of organic nitrogen source. Both yeast extract and casamino acid supplementation enhanced decolorization of direct violet and methyl red dyes and production of lignin peroxidase by the fungal strains. In contrast, the laccases were absent in the similar medium enriched with the same organic nitrogen sources.

Scale translation from shaken to diffused bubble aerated systems for lycopene production by Blakeslea trispora under stimulated conditions.

This study deals with the scale up of Blakeslea trispora culture from the successful surface-aerated shake flasks to dispersed-bubble aerated column reactor for lycopene production in the presence of lycopene cyclase inhibitor 2-methyl imidazole. Controlling the initial volumetric oxygen mass transfer coefficient (kLa) via airflow rate contributes to increasing cell mass and lycopene accumulation. Inhibitor effectiveness seems to decrease in conditions of high cell mass. Optimization of crude soybean oil (CSO), airflow rate, and 2-methyl imidazole was arranged according to central composite statistical design. The optimized levels of factors were 110.5 g/L, 2.3 vvm, and 29.5 mg/L, respectively. At this optimum setting, maximum lycopene yield (256 mg/L) was comparable or even higher to those reported in shake flasks and stirred tank reactor. 2-Methyl imidazole use at levels significantly lower than those reported for other inhibitors in the literature was successful in terms of process selectivity. CSO provides economic benefits to the process through its ability to stimulate lycopene synthesis, as an inexpensive carbon source and oxygen vector at the same time.

Antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of Markhamia tomentosa.

CONTEXT: Plants harbor endophytes with potential bioactivity. Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae) is reported to possess antioxidant, anti-inflammatory and anticancer activities. OBJECTIVE: The antifungal and antiproliferative properties of endophytic fungi extracts and fractions from M. tomentosa were evaluated. MATERIAL AND METHODS: Endophytic fungi were isolated from the leaves of M. tomentosa and identified by ITS-rDNA sequence analysis. The antagonistic effect of the fungal strains was investigated against pathogenic fungi viz, Fusarium oxysporum, Sclerotinia sclerotiorium, Rhizoctonia solani, and Botrytis cinerea using the dual culture assay for 5-7 days. Antiproliferative effect of the fungal extracts and fractions (3.91-250 µg/mL) on HeLa cancer cell line was tested and IC50 was calculated. Poisoning food assay and antifeedant activity against the pathogenic fungi and Spodoptera litura larvae, for 7 days and 2 h, respectively, was also tested at concentrations of 250, 500 and 1000 µg/mL. RESULTS: Fungal endophytes Trichoderma longibrachiatum and Syncephalastrum racemosum were isolated from the leaves of M. tomentosa. Isolated endophytic fungal strains and solvent extracts showed MIC value of 1000 µg/mL against tested pathogenic fungi in the dual culture and poisoning food assays. Methanol fraction of S. racemosum isolate showed the most effective antiproliferative activity with IC50 of 43.56 µg/mL. Minimal feeding deterrent activity against S. litura larvae was also observed. DISCUSSION AND CONCLUSION: These findings showed that the leaves of Markhamia tomentosa harbor strains of endophytic fungi with promising health benefits, and suggest their antifungal and antiproliferative effects against pathogenic fungi and HeLa cancer cell line.

Waste cooking oil: A new substrate for carotene production by Blakeslea trispora in submerged fermentation.

The objective of this study was to evaluate a waste, waste cooking oil (WCO) as substrate for carotene production by Blakeslea trispora in shake flask culture. WCO was found to be a useful substrate for carotene production. B. trispora formed only pellets during fermentation. The oxidative stress in B. trispora induced by hydroperoxides and BHT as evidenced by increase of the specific activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly the production of carotenes. The highest concentration of carotenes (2021 ± 75 mg/l or 49.3 ± 0.2 mg/g dry biomass) was obtained in culture grown in WCO (50.0 g/l) supplemented with CSL (80.0 g/l) and BHT (4.0 g/l). In this case the carotenes produced consisted of ß-carotene (74.2%), γ-carotene (23.2%), and lycopene (2.6%). The external addition in the above medium glucose, Span 80, yeast extract, casein acid hydrolysate, l-asparagine, thiamine. HCl, KH2PO4, and MgSO4·7H2O did not improve the production of carotenes.

Feasibility of raw glycerol conversion into single cell oil by zygomycetes under non-aseptic conditions.

The use of plant oils as feedstock for the biodiesel manufacture has many drawbacks, thus, the interest has turned to single cell oil (SCO) as an alternative. However, the production of SCO is still too expensive, mainly due to the low oil productivity and the high cost of medium sterilization required. In this work raw glycerol was converted into SCO by oleaginous Zygomycetes under non-aseptic conditions on selective (i.e., containing essential oils and/or antibiotics) nitrogen limited media. The obtained data showed that although bacterial populations inhibited the fungal growth, lipid accumulation remained unaffected by the presence of bacteria in the growth medium compared to control experiments (conducted under aseptic conditions). Therefore, a two-stage process was developed in which growth was performed under aseptic conditions (1st stage) followed by lipid accumulation performed under non-aseptic conditions (2nd stage) in the presence of thyme essential oil as an antibacterial agent. Large amounts of lipids were accumulated inside the mycelia, yielding around 13% wt/wt of oil per glycerol consumed.

From cheese whey to carotenes by Blakeslea trispora in a bubble column reactor.

The effect of the aeration rate on carotene production from deproteinized hydrolyzed whey by Blakeslea trispora in a bubble column reactor was investigated. Aeration rate significantly affected carotene concentration and morphology of the fungus. Enhanced aeration caused change of the morphology of B. trispora from pellets with large projected area to pellets with small projected area. This morphological differentiation of the fungus was associated with a significant increase in carotene production. When deproteinized hydrolyzed whey was supplemented with 30 g/l Tween 80, 30 g/l Span 80, and 0.2 % (v/v) ß-ionone, the highest carotene productivity (55.5 mg/g dry biomass/day or 405.0 mg/l/day) was obtained at an aeration rate of 4 vvm. This is the highest carotene productivity that has been reported among the agro-industrial by-products up to date. In this case, the carotenes produced consisted of ß-carotene (67 %), γ-carotene (15 %), and lycopene (18 %).

Enhanced lycopene content in Blakeslea trispora by effective mutation-screening method.

The zygomycete fungus Blakeslea trispora is usually used as a natural source of lycopene and ß-carotene. In this study, the B. trispora (-) strain, a major mating type for lycopene production, was treated with N(+) ion implantation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid (CTA). After several rounds of screening, four mutants with higher yield of lycopene and biomass were isolated. Among these mutants, I5 obtained with N(+) ion implantation showed a maximum lycopene yield (28.8 mg/g), which was 64 % higher than the parent strain (17.5 mg/g) in the production of lycopene. The results indicated that N(+) ion implantation is more suitable for B. trispora (-) than NTG treatment, and the addition of lovastatin promoted the generation of positive mutant and CTA amplified the color differences between colonies.

[Iron and invasive fungal infection]. / Hierro e infección fúngica invasiva.

Iron is an essential factor for both the growth and virulence of most of microorganisms. As a part of the innate (or nutritional) immune system, mammals have developed different mechanisms to store and transport this element in order to limit free iron bioavailability. To survive in this hostile environment, pathogenic fungi have specific uptake systems for host iron sources, one of the most important of which is based on the synthesis of siderophores-soluble, low-molecular-mass, high-affinity iron chelators. The increase in free iron that results from iron-overload conditions is a well-established risk factor for invasive fungal infection (IFI) such as mucormycosis or aspergillosis. Therefore, iron chelation may be an appealing therapeutic option for these infections. Nevertheless, deferoxamine -the first approved iron chelator- paradoxically increases the incidence of IFI, as it serves as a xeno-siderophore to Mucorales. On the contrary, the new oral iron chelators (deferiprone and deferasirox) have shown to exert a deleterious effect on fungal growth both in vitro and in animal models. The present review focuses on the role of iron metabolism in the pathogenesis of IFI and summarises the preclinical data, as well as the limited clinical experience so far, in the use of new iron chelators as treatment for mucormycosis and invasive aspergillosis.

Biotransformation of ursolic acid by Syncephalastrum racemosum CGMCC 3.2500 and anti-HCV activity.

Microbial transformation of ursolic acid (UA, 3ß-hydroxy-urs-12-en-28-oic acid, 1) by filamentous fungus Syncephalastrum racemosum CGMCC 3.2500 was conducted. Five metabolites 3ß, 7ß, 21ß-trihydroxy-urs-12-en-28-oic acid (2); 3ß, 21ß-dihydroxy-urs-11-en-28-oic acid-13-lactone (3); 1ß, 3ß, 21ß-trihydroxy-urs-12-en-28-oic acid (4); 3ß, 7ß, 21ß-trihydroxy-urs-1-en-28-oic acid-13-lactone (5); and 21-oxo-1ß, 3ß-dihydroxy-urs-12-en-28-oic acid (6) were afforded. Elucidation of the structures of these metabolites was primarily based on 1D and 2D NMR and HR-MS data. Metabolite 2 was a new compound. In addition, the anti-HCV activity of compounds 1-6 was evaluated.

Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata.

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3ß-hydroxy-urs-11-en-28,13-lactone (2), 3ß,7ß-dihydroxy-urs-11-en-28,13-lactone (3), 1ß,3ß-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.

Production and derivate composition of trisporoids in extended fermentation of Blakeslea trispora.

Trisporic acid, its precursors and derivatives are used within zygomycete fungi as communication signals and sexual regulators, and also influence the production rate of the parent compound, beta-carotene. Cultivation parameters during growth and the trisporoid production phase of Blakeslea trispora were studied in two-step shake flask cultures and up-scaled fermentations. Comparison of various fermentation protocols allowed the definition of parameters governing trisporoid production. Highest yields were obtained when the initial growth phase allowed for both rapid growth and fast exhaustion of nitrogen and phosporous sources. Onset of trisporoid production is accompanied by a pH drop in the medium and triggered by nutrient limitation, nitrogen depletion being the most important factor. Supplementation of cultures with carbon at low concentration after onset of trisporoid production led to prolonged growth and higher final product accumulation. B. trispora produces trisporoids in two major series, B and C. During a first peak in trisporic acid accumulation, production of trisporic acid B exceeds that of trisporic acid C, which later accumulates at the expense of the trisporic acid B, indicating a variable regulation of the ratio between these metabolites. These data are valuable for tailoring production systems for enrichment of specific intermediates of this complex signal family.

In vitro and in vivo activities of posaconazole against zygomycetes with various degrees of susceptibility.

OBJECTIVES: We analysed the in vitro and in vivo effects of posaconazole and amphotericin B against three clinical isolates of zygomycetes: Lichtheimia corymbifera, F1; and Rhizopus oryzae, F5 and F6. METHODS: In vitro activities of both drugs were assessed by determining MICs, minimum fungicidal concentrations (MFCs) and fungal damage measured by the XTT assay against either the spores or the hyphal forms. Additionally, the survival curves of neutropenic mice systemically infected with the zygomycete isolates were used as the marker of antifungal response to amphotericin B (1 mg/kg/day) or posaconazole (2.5, 10 and 50 mg/kg/day). RESULTS: In terms of MICs, posaconazole proved to be active against the three isolates (MICs ranging from 0.125 to 1.0 mg/L). The median posaconazole MFCs were 0.25, 0.5 and >16 mg/L for F1, F5 and F6, respectively. The XTT assay showed that posaconazole was active against spores of all three isolates, but only partially effective against the hyphae. The survival studies showed that amphotericin B at 1 mg/kg/day and posaconazole at 10 mg/kg/day prolonged the survival of the animals infected with L. corymbifera F1. In mice infected with R. oryzae F5, only posaconazole at 50 mg/kg/day significantly prolonged survival, whereas amphotericin B at 1 mg/kg/day was the only regimen active against R. oryzae F6. CONCLUSIONS: Our findings showed that posaconazole could be useful in the treatment of zygomycosis. Also, we report that an isolate of R. oryzae with low MFC responded to posaconazole, while another isolate with high MFC did not.

[Microbiological transformation of paeoniflorin and albiflorin].

OBJECTIVE: To investigate the microbiological transformation of paeoniflorin and albiflorin. METHOD: The bacteria strains able to transform paeoniflorin and albiflorin were screened from 18 strains of microorganisms. The products were isolated by chromatography method and their structures were elucidated by spectral technology. RESULT: It was found that Cunninghamella blakesleana (AS 3.970) and Syncephalastrum racemosum (AS 3.264) could convert paeoniflorin and albiflorin efficiently, respectively. C. blakesleana could convert paeoniflorin to produce albiflorin, while S. racemosum could convert albiflorin to produce paeoniflorin. CONCLUSION: Paeoniflorin and albiflorin could be converted each other in definited condition.

Oxidative stress response and morphological changes of Blakeslea trispora induced by butylated hydroxytoluene during carotene production.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by butylated hydroxytoluene (BHT) during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. The addition of exogenous BHT to the medium caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by remarkable increase of the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a fivefold increase of carotene production. The highest concentration of carotenes (125.0 mg/g dry biomass) was obtained in culture grown in medium supplemented with 20 mM of BHT.

Identification of carotenoids produced from cheese whey by Blakeslea trispora in submerged fermentation.

The identification of carotenoids in B. trispora during pigment production from deproteinized hydrolyzed whey supplemented with plant oils was studied. The carotenoid content in Blakeslea trispora were beta-carotene, gamma-carotene, and lycopene. The composition of carotenoids depends of the amount of oils added to the cheese whey. At the maximum concentration of carotenoids, the proportions of beta-carotene, gamma-carotene, and lycopene (as percent of total carotenoids) was 60.1%, 32.5%, and 7.4%, respectively.

The role of iron and iron chelators in zygomycosis.

Iron is an essential element for cell growth and development, contributing to DNA synthesis and regulating the G(1)-phase to S-phase transition. Moreover, iron is important for the virulence of the majority of microorganisms, and the function of the genes regulating iron uptake is coupled with the manifestations of the virulence phenotype. All fungi elaborate specific uptake mechanisms to sequester iron, and most commonly produce small molecules with high affinity for iron, the siderophores. The importance of iron appears to be particularly high for Zygomycetes, which grow abundantly in iron-rich media, and all the known predisposing factors for zygomycosis have, as a common feature, the increased availability of free iron. Among the known iron chelators, deferoxamine supports the growth of Zygomycetes because it acts as xenosiderophore, delivering iron to iron-uptaking molecules of these species. Conversely, the newer iron chelators deferiprone and deferasirox do not exhibit similar activity, apparently because they share higher affinity constants for iron and, as a result, deprive the fungi of iron, inhibiting their growth. This activity has been documented in various culture systems and in many animal models of zygomycosis, and therefore suggests that these drugs might be used as adjuvant treatment for systemic zygomycosis. There are few case reports in which the newer iron chelators have been used as antifungals, and their possible benefit must be verified in a prospective randomized trial.

Protein enrichment of apple pomace and use in feed for Nile tilapia.

The purpose of this paper is to investigate the protein enrichment of apple pomace by Gongronella butleri through solid-state cultivation and addition of this material as feed for tilapia fry (Oreochromis niloticus). Factorial experimental design was used for the assessment of culture conditions to determine the effects of the source of nitrogen, initial moisture, and granulometry on the protein enrichment of apple pomace. During culture, the consumption of reducing sugars and the production of soluble protein were determined. The best conditions obtained were with urea (5% w/w), initial moisture of 70% and granulometry in the range from 0.85 to 1.70 mm, producing 19.63% of soluble protein. The fry submitted to the diet containing treated apple pomace presented an increase of 44% in body mass, demonstrating that apple pomace biotransformed may represent an excellent food supplement.

Iron acquisition: a novel perspective on mucormycosis pathogenesis and treatment.

PURPOSE OF REVIEW: Mucormycosis is an increasingly common fungal infection with an unacceptably high mortality despite first-line antifungal therapy. Iron acquisition is a critical step in the causative organisms' pathogenetic mechanism. Therefore, abrogation of fungal iron acquisition is a promising therapeutic strategy to impact clinical outcomes for this deadly disease. RECENT FINDINGS: The increased risk of mucormycosis in patients with renal failure receiving deferoxamine iron chelation therapy is explained by the fact that deferoxamine actually acts as a siderophore for the agents of mucormycosis, supplying previously unavailable iron to the fungi. The iron liberated from deferoxamine is likely transported into the fungus by the high-affinity iron permease. In contrast, two other iron chelators, deferiprone and deferasirox, do not supply iron to the fungus and were shown to be cidal against Zygomycetes in vitro. Further, both iron chelators were shown to effectively treat mucormycosis in animal models, and one has been successfully used as salvage therapy for a patient with rhinocerebral mucormycosis. SUMMARY: Further investigation and development of iron chelators as adjunctive therapy for mucormycosis is warranted.