The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1β, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

No abstract

Cottonseed cake biomass, which is a residue of oil extraction, is potentially appropriate for use as animal feed, given the high mineral, fibre and protein content. The presence of free gossypol, however, a toxic pigment in the glands of the cotton plant, limits use of this biomass for monogastric livestock. A promising method to detoxify cottonseed cake relies on fermentation by fungi, which can eliminate up to 100% of gossypol. In order to quantify trace levels of free gossypol in different cotton materials, including cottonseed cake treated with macrofungi, a simple and rapid chromatographic detection method was developed and validated. Under optimized conditions, extraction was performed using 70% acetone. The extract was then analysed by Ultra High-Performance Liquid Chromatography (UHPLC), with gradient elution on a C18 reverse phase column KINETEX® (100 x 2.10 mm, 2.6 μm). Methanol-0.1% TFA aqueous solution was employed as mobile phase and PDA detection conducted at 254 nm. The optimized method was validated by analysis of specificity, linearity and range, limit of detection, limit of quantification, precision and accuracy. Detection and quantification limits were observed at 0.2 and 0.5 μg/mL, respectively. With good reproducibility, with precision (RSD)<10% and recovery greater than 94%, the developed assay was appropriate for quantification of low quantities of free gossypol. The validated method was successfully applied to determine trace levels of free gossypol cottonseed treated with a macrofungus.

No abstract

No abstract

Fungi can produce many bioactive metabolites, which are enhanced when challenged in co-culture competition. For a better evaluation of these metabolites, Mass Spectrometry Imaging (MSI) can be used to provide complementary information about the metabolite spatial localization. However, some adaptations are required on available methodologies in MSI for applications in microorganisms, particularly on sample preparation, due to the characteristics of each type of cell that has to be analyzed. The imprinting method has been shown to be a robust method when applied to sample preparation, but to our knowledge it has never been tested for microbial MALDI-MSI. Herein we applied both Classic and Imprinting MALDI-MSI to compare and analyze metabolites produced by Aspergillus terreus (ATCC® 20542TM) and Pleurotus pulmonarius fungi. For the classic method, the fungi were inoculated for 8 days with PDA medium in a MALDI glass slide. For the imprinting method, fungi were also inoculated for 8 days in a MALDI glass slide and then transferred to a filter paper by manual pressure using a homemade apparatus. Samples were then dehydrated and submitted to HCCA matrix application by sublimation. The chemical images were acquired by MALDI-MSI, and the metabolites were identified by UHPLC-ESI-MS/MS. Twelve ions were detected by MALDI-MSI, using classic (m/z 210.54, 276.99, 307.45, 321.04, 329.70, 346.12, 351.12, 462.41 and 484.02) and imprinting (m/z 313.64, 379.66 and 404.36) methods. Some ions presented a higher intensity in the interaction zone between fungi areas, especially the ions m/z 329.70, 351.12 and 484.02. These ions may be related to metabolites involved in communication between microorganisms, because these fungi formed a mutualistic interaction. All ions were investigated by UHPLC-ESI-MS/MS, and two were identified: adenosine monophosphate (C10H14N5O7P, m/z 346.12, [M-H]-) visualized in the Classic Method, and rubrophen (C22H20O6, m/z 379.66, [M-H]-) visualized in the Imprinting Method. The metabolites from microorganisms are rarely reported in MS/MS databases, which explains the difficulty in the identification of these compounds. Our MSI analysis using the classic method provided a higher number of detected ions. However, both classic and imprinting methods resulted in a complementary information, leading to the detection of ions that were not previously observed on the classic approach. Despite of the challenges encountered on the sample preparation and metabolite identification, using both classic and imprinting MALDI-MSI for bioprospection of fungi metabolites is a promising approach on the analytical field of mass spectrometry which can be later used in biotechnological applications.

No abstract

Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C and xylanase was reduced in 53% after two hours incubation at the same temperature. CMCase and xylanase obtained by A. niger KIJH cultivated in submerged culture containing J. corumbensis as carbon source may have application in biotechnology processes that require enzymes that remain active under routine extreme conditions.