Degradation of Aflatoxin B1 by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of Pleurotus eryngii

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g−1 dry matter) and low MnP activity (0.4 U g−1 dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable

In Vitro Downregulation of Matrix Metalloproteinase-9 in Rat Glial Cells by CCR5 Antagonist Maraviroc: Therapeutic Implication for HIV Brain Infection

BACKGROUND: Matrix metalloproteinases (MMPs) released by glial cells are important mediators of neuroinflammation and neurologic damage in HIV infection. The use of antiretroviral drugs able to combat the detrimental effect of chronic inflammation and target the exaggerated MMP activity might represent an attractive therapeutic challenge. Recent studies suggest that CCR5 antagonist maraviroc (MVC) exerts immunomodulant and anti-inflammatory activity beyond its anti-HIV properties. We investigated the in vitro effect of MVC on the activity of MMPs in astrocyte and microglia cultures. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of rat astrocytes and microglia were activated by exposure to phorbol myristate acetate (PMA) or lypopolysaccharide (LPS) and treated in vitro with MVC. Culture supernatants were subjected to gelatin zymography and quantitative determination of MMP-9 and MMP-2 was done by computerized scanning densitometry. MMP-9 levels were significantly elevated in culture supernatants from both LPS- and PMA-activated astrocytes and microglia in comparison to controls. The treatment with MVC significantly inhibited in a dose-dependent manner the levels and expression of MMP-9 in PMA-activated astrocytes (p<0,05) and, to a lesser extent, in PMA-activated microglia. By contrast, levels of MMP-2 did not significantly change, although a tendency to decrease was seen in PMA-activated astrocytes after treatment with MVC. The inhibition of levels and expression of MMP-9 in PMA-activated glial cells did not depend on cytotoxic effects of MVC. No inhibition of MMP-9 and MMP-2 were found in both LPS-activated astrocytes and microglia. CONCLUSIONS: The present in vitro study suggests that CCR5 antagonist compounds, through their ability to inhibit MMP-9 expression and levels, might have a great potential for the treatment of HIV-associated neurologic damage

Bioremediation of aflatoxin B1-contaminated maize by king oyster mushroom (Pleurotus eryngii).

Aflatoxin B1 (AFB1) is the most harmful mycotoxin that occurs as natural contaminant of agricultural commodities, particularly maize. Practical solutions for detoxification of contaminated staples and reduction of agricultural wastes are scarce. We investigated the capability of the white-rot and edible fungus Plerotus eryngii (king oyster mushroom) to degrade AFB1 both in vitro and in a laboratory-scale mushroom cultivation, using a substrate similar to that routinely used in mushroom farms. In malt extract broth, degradation of AFB1 (500 ng/mL) by nine isolates of P. eryngii ranged from 81 to 99% after 10 days growth, and reached 100% for all isolates after 30 days. The growth of P. eryngii on solid medium (malt extract-agar, MEA) was significantly reduced at concentrations of AFB1 500 ng/mL or higher. However, the addition of 5% wheat straw to the culture medium increased the tolerance of P. eryngii to AFB1 and no inhibition was observed at a AFB1 content of 500 ng/mL; degradation of AFB1 in MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour was 71-94% after 30 days of growth. Further, AFB1 degradation by P. eryngii strain ITEM 13681 was tested in a laboratory-scale mushroom cultivation. The mushroom growth medium contained 25% (w/w) of maize spiked with AFB1 to the final content of 128 μg/kg. Pleurotus eryngii degraded up to 86% of the AFB1 in 28 days, with no significant reduction of either biological efficiency or mushroom yield. Neither the biomass produced on the mushroom substrate nor the mature basidiocarps contained detectable levels of AFB1 or its metabolite aflatoxicol, thus ruling out the translocation of these toxins through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1- contaminated corn through the exploitation of the degradative capability of P. eryngii and its bioconversion into high nutritional value material intended for feed production

Inhibitory effect of AFB₁ on growth of <i>P</i>. <i>eryngii</i>.

<p>The isolates ITEM 13681, ITEM 13688 and ITEM 13697 were grown for 15 days at 30 ± 1°C in the dark on malt extract agar (MEA) containing different concentrations (60, 120, 250, 500, 1000 ng/mL) of AFB₁. Data are the means ± SD (n = 5) of the percent reduction in colony diameters with respect to control. Statistically significant differences with control are indicated by asterisks (*** = <i>P</i> < 0.001, ** = <i>P</i><0.01; One-way Anova).</p

Determination of AFOL in the biomass of <i>P</i>. <i>eryngii</i>.

<p>Overlay of HPLC/FLD chromatograms of a standard solution of aflatoxicol (AFOL, black line) and the extract of <i>P</i>. <i>eryngii</i> biomass developed on the contaminated mushroom substrate (red line). AFOL was not present in the extract.</p

Lead exposure of rats during and after pregnancy induces anti-myelin proteolytic activity: a potential mechanism for lead-induced neurotoxicity

Toxic effects of lead (Pb) are principally manifested in the central nervous system (CNS) and a mounting body of evidence indicates that excessive chronic exposure to Pb participates in the pathological processes of numerous neurodegenerative disorders in humans.In this study we evaluated whether the prolonged pre- and postnatal exposure of rat pups to lead, administrated through ingestion in drinking water, as a typical environmental exposure, can determine alterations of the protein pattern of CNS myelin and the induction of myelin-associated proteinases. Pregnant dams were given distilled water or 0.3 mg/mL lead acetate in drinking water during gestation and lactation. At postnatal day (PND) 21, pups born from mothers poisoned with Pb continued the treatment with the metal. On PND 35 and 56, pups were sacrificed, and brains were subjected to myelin purification and extraction of myelin-associated proteinases. The SDS-PAGE analysis of protein pattern of myelin incubated in vitro with an oxidative system indicated that myelin proteins from Pb-treated pups were more sensitive to the toxicity of reactive oxygen species in comparison with those from untreated pups. The zymografic analysis of NaCl-extracts from myelin of Pb-treated pups showed a band of digestion of 54 kDa that increased in pups sacrificed at PND 56 in comparison with those sacrificed at PND 35 and correlated with the concentration of Pb, detected in purified myelin. The incubation of the NaCl-extract from Pb-treated pups with purified myelin basic protein (MBP) evidenced the presence of different MBP-degrading activities. These results suggest that Pb may influence the integrity of the myelin sheath, probably through the induction of anti-myelin proteinases

Potential of <i>Trichoderma</i> spp. for Biocontrol of Aflatoxin-Producing <i>Aspergillus flavus</i>

The inhibitory action of 20 antagonistic Trichoderma isolates against the aflatoxigenic isolate A. flavus ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites with inhibitory effect by the Trichoderma isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B1 (AfB1) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB1. Reduction of AfB1 content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of Trichoderma was not necessarily associated with inhibition of AfB1 production and vice versa, we investigated the mechanism of reduction of AfB1 content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes aflR and aflS, and the structural genes aflA, aflD, aflO and aflQ of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB1 content reduction by these Trichoderma strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of A. flavus and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions, Trichoderma spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use