Optimization of process variables for supercritical fluid extraction of ergothioneine and polyphenols from Pleurotus ostreatus and correlation to free-radical scavenging activity

Supercritical carbon dioxide extraction was employed to extract antioxidants from Pleurotus ostreatus. The response surface methodology was employed to determine the optimal conditions for extraction of ergothioneine and polyphenols. The ergothioneine concentration in the mushroom extract was quantified and characterized using high pressure liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The optimized values of responses were obtained at a pressure of 21 MPa, a temperature of 48 °C and a co-solvent amount of 133 ml, yielding an ergothioneine content of 1.35 mg/g dw, total phenol content of 5.48 mg GAE/g dw, and IC50 for DPPH radical scavenging capacity of 0.008 mg/ml. A higher desirability value of 0.98 for model demonstrated that response surface methodology can be successfully applied for optimizing supercritical carbon dioxide extraction of antioxidants from P. ostreatus. A good correlation was found between DPPH radical scavenging capacity and ergothioneine (R² = 0.94) as well as with polyphenols (R² = 0.95).     

Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity

Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii and Grifola frondosa were analyzed for antioxidant capacity, as measured by oxygen radical absorbance capacity (lipophilic and hydrophilic) (ORAC_total), hydroxyl radical averting capacity (HORAC), peroxynitrite radical averting capacity (NORAC), superoxide radical averting capacity (SORAC) assays, Folin-Ciocalteu reagent and ergothioneine (ERG) content. ORAC_totalvalues ranged from 39 to 138 μmol of Trolox equivalents (TE)/g dry weight (dw). HORAC values ranged from 3.0 to 13.6 μmol of caffeic acid equivalents/g dw. NORAC values ranged from 2.0 to 9.0 μmol TE/g dw. SORAC values ranged from 0.37 to 2.6 kunit superoxide dismutate equivalents/g dw. Polyphenols ranged from 4.2 to 10.6 mg gallic acid equivalents/g dw. A. bisporus mushrooms, especially portabellas, had higher antioxidant capacity relative to the specialty mushrooms tested. ERG ranged from 0.21–2.6 mg/g dw with L. edodes, P. ostreatus, G. frondosa containing a statistically significant greater amount compared to A. bisporus. A good correlation was found between ORAC_total and polyphenols (R² = 0.86).     

Mycobial enhancement of ergothioneine by submerged cultivation of edible mushroom mycelia and its application as an antioxidative compound

Ergothioneine (2-mercaptohistidine trimethylbetaine, ESH) in mushroom is one of bioactive and functional components. In the present study, a method of producing ESH within a short period and with high productivity was established by applying submerged fermentation of edible Shiitake mushroom (Lentinula edodes) mycelia. High-resolution mass spectrometry and online flow injection analysis of 2,2-diphenyl-1-picrylhydrazyl scavenging activity clearly confirmed that ESH was the most potent antioxidant in the mycelial extract. Among several mushroom mycelia, Shiitake produced the highest amount of ESH (0.60 mg/g dry weight (DW)). Optimisation of the culture medium was performed to achieve higher ESH production. The monosaccharide content in the medium, particularly fructose in combination with aspartic acid, was one of the factors that enhanced ESH production by 3.15-fold (1.89 mg/g DW). Individual supplementation of 2 mM methionine as an amino acid precursor yielded a significantly higher amount of ESH (3.45 mg/g DW) compared to that of the control medium (1.85 mg/g DW). The mycelial extracts containing ESH were effective in retarding the formation of secondary lipid oxidation products in the yellowtail red meat model during chilled storage as well as in the oxidation of linoleate and bleaching of beta-carotene in an oil-in-water emulsion system.     

Biochemical intervention of ergothioneine-rich edible mushroom (Flammulina velutipes) extract inhibits melanosis in crab (Chionoecetes japonicus)

We investigated the effects of decreasing phenoloxidase (PO) activity and prophenoloxidase (proPO) gene expression on the inhibition of postharvest melanosis formation in the red queen crab, Chionoecetes japonicus. The cDNA of proPO from hemocytes of C. japonicus was partially cloned and sequenced. Immersion of live crabs in a 1.0% ergothioneine (ESH)-rich mushroom extract (Flammulina velutipes; ME) solution resulted in significant inhibition of haemolymph PO activity and a reduction of the proPO gene expression in hemocytes that consequently controlled melanosis in the crabs during ice storage. Treatments with a 0.05% w/v sodium sulphite solution or a 0.05% w/v 4-hexyl-1,3-benzenediol solution had similar positive effects as the treatment with a 1.0% ME solution in vivo. In vitro experiments showed that authentic l-(+)-ESH inhibited PO activity and decreased proPO gene expression in crab hemocytes. Thus, the application of ESH-rich ME can be a novel alternative to synthetic melanosis-inhibiting agents to control postharvest melanosis in crabs.     

A rapid HPLC post-column reaction analysis for the quantification of ergothioneine in edible mushrooms and in animals fed a diet supplemented with extracts from the processing waste of cultivated mushrooms

For establishing an efficient and sensitive method for the quantitative determination of 2-thiol-l-histidine-betaine (ergothioneine, ERG) in edible mushrooms and the blood and muscles of animals, a technique using reversed-phase separation and post-column reaction between 2′-dipyridyl disulphide and ERG was developed. A corresponding derivative 2-thiopyridone, detected at 343 nm, was used for estimating ERG concentration. The flow rate, temperature, pH, and composition of the solution were optimised. A low limit of quantification (1.41 ppm) and a simpler sample preparation made this technique more rapid compared to other methods using liquid chromatography–mass spectrometry. The coefficient of variation (CV) values for the reproducibility and recovery of ERG were within the acceptable values of 6% and 97.5–100.0%, respectively. The efficiency of this methodology was compared with that of spectrophotometric and mass-spectrometric quantitative methods, and was assessed in the light of previous studies. The ERG contents in different mushrooms were 12.69–234.85 mg/kg wet weight basis. Dietary supplementation with extracts from mushroom processing waste significantly improved ERG bioavailability in the blood of yellowtail fish and muscle tissue of cattle.