Studies on Biofortification of Selenium in Edible Mushrooms

Abstract

Seven edible mushroom strains namely Agaricus bisporus (U-3) and Volvariella volvacea (Vv-4) and five strains of Pleurotus, viz. Pleurotus florida (DMRP-136), Pleurotus djamor (DMRP-205), Pleurotus sajorkaju (DMRP-112), Pleurotus citrinopileatus (DMRP-10) and Pleurotus ostreatus (DMRP-2) were cultivated on agricultural residues belonging to selenium rich and non enriched sites. All Se-rich strains were found to accumulate significantly high selenium content than respective non-Se strains, wherein Se-rich P. djamor (145.4 µg Se/gDW) was found to accumulate relatively higher total selenium content than other mushroom species and Se-rich P. citrinopileatus (26.1 µg Se/gDW) accumulated the lowest. Total soluble form of selenium in gastrointestinal extracts (GI) of P. florida as analysed by ICP-DRC-MS was found to be 105.8 ± 1.6 μg/g dw, which provided an indication that 75% of selenium was appreciably bioavailable to the body. Investigation of the Se molecular fractions revealed that proteins and other high molecular weight selenium-containing molecule were hydrolyzed to peptides and low molecular weight selenocompounds (≤5 kDa based on column calibration). Se speciation in the GI was dominated by selenomethioneine, which represented 73% of the sum of the detected species. Only 2% of the detected species was present as inorganic Se in the form of Se (IV). A number of other low molecular weight selenocompounds generated during the gastrointestinal digestion, accounting for 25% of the chromatographed Se, remained unidentified. Mushrooms cultivated on naturally Se-enriched substrates did not differ from controls in terms of yield and biological efficiency. Mushroom cultivated on Se-rich agricultural residues were examined for their elemental content with respect to their control. In general, the CHN for selected mushrooms (P. florida, P. sajorkaju, P. djamor, P. ostreatus and P. citrinopileatus) and Fe, Zn, Cu, Mg, Ca content for all seven species (A. bisporus, V. volvacea, P. florida, P. sajorkaju, P. djamor, P. ostreatus and P. citrinopileatus) was found to be similar in fruiting bodies of both selenium enriched and control mushrooms. Se-rich fruiting bodies of all mushrooms have shown significantly higher antioxidant status in terms of all antioxidant assays as compared to control ones. However, when compared at species level, different species have shown varying levels of selenium uptake and therefore varying antioxidant activity. Se-rich V. volvacea (17.7 ± 0.5 mg GAE/ g DW) showed highest phenol content, Se-rich A. bisporus has shown maximum lipid peroxide inhibition (17.6 ± 4.4 nM MDA/g) and highest free radical scavenging (91.4 ± 1.4 % at 10 mg/ml) with EC50 value of 0.056 mg/ml as compared to other Se-enriched species respectively. Similarly, Se-rich P. sajorkaju exhibited relatively higher metal chelation with EC50 value of 2.12 mg/ml as compared to other Se and NSe species. To examine the effect of bioactive fractions of selected species (A. bisporus, V. volvacea and P. sajorkaju) on proliferation of A549 lung cancer cells, the crude extracts of mushrooms (Se and NSe) from four different solvents viz. hexane, ethanol, methanol and water were tested. Se-rich bioactive extracts showed significantly higher cytotoxicity than NSe extracts against the cell lines. The Se-rich fraction inhibited the proliferation by 89.6%, 87.9%, 64.9% and 47.5% as compared to 83.2%, 42.3%, 4.4% and 9.4% inhibition by non-Se mushrooms in hexane, ethanol, methanol and water fractions respectively. In case of V. volvacea, various solvent fractions showed marginal to significant inhibition of cell proliferation. The Se-rich fraction inhibited the proliferation by 98.87%, 74.8%, 66.0% and 54.0% as compared to 98.71%, 70.5%, 55.0% and 3.0% inhibition by non-Se mushrooms in water, hexane, ethanol and methanol fractions respectively. Similarly, in case of oyster mushroom (P. sajorkaju), ethanol fraction showed maximum inhibition of cell proliferation followed by methanol, water and hexane. The Se-rich fraction inhibited the proliferation by 91.8%, 88.3%, 83.0% and 71.6% as compared to 0%, 11.1%, 55.4% and 51.0% inhibition by non-Se mushrooms in ethanol, methanol, water and hexane fractions respectively. However, when compared at species level among the three different mushroom species; Se-rich bioactive extracts of P. sajorkaju showed much potential in inhibiting proliferation of cancer cells in all four fractions. The Se-rich extracts were analyzed for possible presence of selenium analogue of ergothioneine (selenoergothioneine like moiety) using FTIR and LC-MS. A preliminary indication of presence of selenoergothioneine like moiety, was reached by FTIR wherein the characteristic peak at 931 cm−1 indicated the existence of C=Se in the structure. The presence of selenoergothioneine was furthur indicated through results of LC-MS analysis, with a molecular ion [M+H]+ peak at m/z 278.1 corresponding to the molecular formula C9H15N3O2Se and calculated mass of 277.1. Essentiality of selenium as a micronutrient is gaining ground extensively. A significant component of world’s population is at risk of exposure to several diseases due to sub-optimal selenium intake. Therefore the study envisages and proposes the use of Se-hyperaccmulated mushrooms with enhanced bioactivity for preparation of Se supplemented neutrceuticles that can facilitate bioavaliable and accessible Se required to overcome the deficiency. As an added benefit, this approach can also facilitate the re-use of Se-rich agricultural residues which otherwise cause environmental hazard due to burning of post-harvest residues in the region.