Analysis of mushroom composition

Around one kilogram of complete (pileus and stipe) and mature fruiting bodies of each experimental treatment were needed to perform the different tests of composition. The different tests were performed on fresh samples, and then results were provided in percentage dry weight by conversion. Total protein content of samples was estimated by the macro-Kjeldahl method (N × 4.38)⁴⁰. Determination of total carbohydrates content followed the Anthrone method³⁶. Fat content was determined by extracting a known weight of powdered sample with ethyl ether, using a Soxhlet apparatus. The percentage of fiber was calculated, based on AOAC official method 962.09⁴¹ general method, after determining the ash content in mushrooms (2 g of mushrooms put in a crucible and set in a muffle furnace at 500 °C for 12 h, then the furnace removed, cooled in a desiccator and weighed). Thereafter, 0.5 g of dried sample were extracted using acetone in a 50 ml centrifuge tube, the extract containing fat was discarded, and 50 ml of H₂SO₄ (1.25%) were added, the mixture was boiled at 100 °C for 15 min, then centrifuged at 1000 rpm, decanted and the solution discarded. This was followed by the addition of 50 ml NaOH (1.25%), boiling at 100 °C for 15 min, centrifugation at 1000 rpm, decantation, discarding again the solution, adding 25 ml H₂SO₄ (1.25%), decanting solution, adding 50 ml H₂O, filtering the solution on a pre-weighed filter paper using Buchner, washing with 2 H₂O portions 50 ml each then using 25 ml ethanol portion. Therefore, the residue was dried for 2 h at 130 °C in oven; the remaining residue represented fiber and ash contents. The percentage of fiber was then calculated (Eq. ¹).

The analysis of amino acid composition followed ISO standards (13903:2005)⁴². Free amino acids were extracted with hydrochloric acid. Co-extracted nitrogenous macromolecules were precipitated with sulfosalicylic acid and removed by filtration. The filtered solution was adjusted to a pH of 2.2. Amino acids were then separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm.

For the measurements of ions (Ca, K, Mn, Fe, Na and Mg), 2.8 ml of HNO₃ (65%) were added to 5–6 g of samples, digested at 150 °C for 1 h, then filtrated with 100 ml of distilled water. The filtrate was then subjected to Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Phosphorus content was determined by spectrophotometric method, wherein phosphorus reacts with molybdic acid to form phosphomolybdate complex, which was then reduced with amino naphthol sulfonic acid to complex molybdenum blue that was measured spectrophotometrically⁴³.

Zinc, copper, nickel and lead were determined using Atomic Absorption Spectrophotometer (AAS) (Perkin Elmer, Model Analyst 400, Waltham, Massachusetts, USA); mushroom samples were digested using a mixture of HNO₃, H₂SO₄, and H₂O₂ (4:1:1) (12 ml per 1 g of sample). The mixture was boiled to 150 °C for 4 h, and then deionized water was added to a volume of 25 ml. Same steps were followed to prepare a blank digest. Standard solutions for calibration were prepared by diluting a stock solution of 1000 mg/l (Sigma and Aldrich) of each tested element.

For the determination of soluble sugars, mushroom samples were heated in water at 100 °C for 30 min. The sample solution was filtered and 20 µl of filtrate were used then for normal phase extraction using High Performance Liquid Chromatography (HPLC) at 30 °C (normal phase extraction; column NH₂ column: 250 × 4.5 mm ID, at a flow rate of 1.2 ml min⁻¹). Sugars were detected with a refractive index detector (RID) (mobile phase: mixture of polar-non-polar solution, calibration: using a 2 points concentration). Sugar identification was made by comparing with standards prepared from stock solution of sugars to get concentrations approximate to sample. For the different tests, each represented value is the mean of 3 replicates determination ± standard deviation.