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

Inhibition of myelin-cleaving poteolytic activities by interferon-beta in rat astrocyte cultures. Comparative analysis between gelatinases and calpain-II.

Proteolytic enzymes have been implicated in the pathogenesis of Multiple Sclerosis (MS) for both their ability to degrade myelin proteins and for their presence in MS plaques.In this study we investigated whether interferon-beta (IFN-β) could differently modulate the activity and the expression of proteolytic activities against myelin basic protein (MBP) present in lipopolysaccharide (LPS)-activated astrocytes.Rat astrocyte cultures were activated with LPS and simultaneously treated with different doses of IFN-β. To assess the presence of MBP-cleaving proteolytic activity, culture supernatants and cellular extracts collected from astrocytes were incubated with exogenous MBP. A MBP-degrading activity was found in both lysates and supernatants from LPS-activated astrocytes and was dose-dependently inhibited by IFN-β. The use of protease inhibitors as well as the zymographic analysis indicated the presence of calpain II (CANP-2) in cell lysates and gelatinases A (MMP-2) and B (MMP-9) in cell supernatants. RT-PCR revealed that the expression of CANP-2 as well as of MMP-2 and MMP-9 was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFN-β treatment. The expression of calpastatin, the natural inhibitor of CANPs, was not affected by IFN-β treatment. By contrast, decreased expression of TIMP-1 and TIMP-2, the natural inhibitors of MMP-9 and MMP-2, respectively, was observed in IFN-β-treated astrocytes compared to LPS-treated cells. The ratio enzyme/inhibitor indicated that the effect of IFN-β treatment is more relevant to CANP-2 than on MMPs.These results suggest that the neuroinflammatory damage during MS involves altered balance between multiple proteases and their inhibitors and indicate that IFN-β is effective in regulating different enzymatic systems involved in MS pathogenesis

Degradation of AFB₁ by <i>P</i>.<i>eryngii</i> grown on malt extract-agar plus wheat straw and maize flour (MEASM) supplemented with 500 ng/mL of AFB₁, after 15 and 30 days of incubation at 30 ± 1°C in dark.

<p>Degradation of AFB₁ by <i>P</i>.<i>eryngii</i> grown on malt extract-agar plus wheat straw and maize flour (MEASM) supplemented with 500 ng/mL of AFB₁, after 15 and 30 days of incubation at 30 ± 1°C in dark.</p

<i>Pleurotus eryngii</i> cultivated in a AFB₁-contaminated mushroom medium.

<p>Production of basidiocarps (fruit bodies) by <i>P</i>.<i>eryngi</i>i ITEM 13681 in a mushroom cultivation medium; A) medium contaminated with 128 μg/kg of AFB₁; B) non-contaminated control. In both the conditions <i>P</i>. <i>eryngii</i> ITEM 13681 produced well-developed basidiocarps, as well as growing immature fruit-bodies and fruit primordials in 42 days.</p

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

Reduction of the inhibitory effect of AFB₁ on <i>P</i>. <i>eryngii</i> in the presence of wheat straw.

<p>The isolates ITEM 13681, ITEM 13688 and ITEM 13697 were grown on malt extract agar (MEA), MEA supplemented with 5% (w/v) wheat straw (MEAS) and MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour (MEASM) containing 500 and 1000 ng/mL of AFB₁. Data are the means ± SD (n = 5) of the percent reduction in colony diameters with respect to controls after 15 days of growth at at 30 ± 1°C in the dark. 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