Winaspect plus software

The laccase activity was determined by using ABTS as substrate and measuring the absorbance change caused by the action of enzyme on the substrate within 1 min using a Specord 200 spectrophotometer (Analytic Jena, Germany) and WinASPECT PLUS software [31 (link)]. Molar absorption coefficient (ε) for ABTS at 420 nm and for DMP at 470 nm was 36,000 M^-1 cm^-1 and 14,800 M^-1 cm^-1 [32 (link)]. The reaction mixture contained 250 μL of 10 mM ABTS or DMP, 740 μL of Mc Ilvaine Buffer (composed of 0.2 M dipotassium hydrogen phosphate and 0.1 M citric acid, buffered to pH 4.5), and 10 μL of enzyme extract (Jasińska et al. [33 (link)]. One unit of laccase activity (U) was defined as the concentration of the enzyme required to oxidize 1 μM of substrate per minute.
Enzyme activity was calculated according to the formula adopted from Leonowicz and Grzywnowicz [34 (link)] with slight modifications:
$\mathrm{L}\mathrm{a}\mathrm{c}\mathrm{c}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left[\mathrm{U}/\mathrm{L}\right]=(\mathrm{\Delta }\mathrm{A}\mathrm{b}\mathrm{s}\times \mathrm{V}\times {10}^6)/\left(36,000\times \mathrm{V}\mathrm{e}\times \mathrm{\Delta }\mathrm{t}\right),$
where ΔAbs is the difference in absorbance values at Abs₆₀ and Abs₀;
V is the total volume of the sample [mL]; Ve is the volume of the enzyme [mL]; and Δt is the time [min].
The protein concentration was determined using the BCA test (bicinchoninic acid assay) according to the Pierce^™ BCA Protein Assay Kit protocol (Thermo Fisher Scientific).

UV-Vis absorbance measurements were performed using a dual-trace spectrometer Specord^® 210 Plus from Analytik Jena AG (Jena, Germany). The instrument was operated using Win Aspect Plus software (Analytik Jena AG, Jena, Germany). For Con A concentration determination, a Spectral Scan from 270–290 nm was used. The concentration of Con A expressed in terms of the monomer (M_w = 26,500 g/mol) was then determined at 280 nm applying the Beer-Lambert law with ε₂₈₀ = 30,150 M⁻¹·cm⁻¹ for tetravalent ConA. For glyco-AuNP concentration determination, a Spectral Scan from 390–410 nm was used. Based on the assumption that the nanoparticles are spheres with a radius of 7 nm and that an absorbance of A = 0.3 at a wavelength of 400 nm corresponds to a gold concentration of c(Au⁰) = 1.25 × 10⁻⁴ M, the concentration of the nanoparticles was determined using the equation described by X. Liu et al. [76 (link)]. All solutions were measured in a 3.5 mL precision quartz glass cuvette (d = 1 cm) from Hellma Analytics (Müllheim, Germany).

The color of the acetone mushroom extracts was measured using the WinASpect Plus software version 4.0.0.0 (Analytik Jena, Jena, Germany) module of the Specord 200 Plus UV-Vis spectrophotometer (Analytik Jena, Jena, Germany) for the analysis of color according to DIN EN ISO 1164 (xyz, CIE, L*a*b) and ASTM E 313 (yellowness and whiteness indices) using the standard illuminant D65, field of view 2°.
The color was expressed as L* (lightness/darkness), a* (red/green) and b* (yellow/blue). The color difference between dried and raw mushrooms (ΔE*) was determined according to the equation: $$\mathsf{\Delta }\mathrm{E}{*}^{}=\sqrt{{\mathsf{\Delta }\mathrm{L}}^{\ast 2}+{\mathsf{\Delta }\mathrm{a}}^{\ast 2}+{\mathsf{\Delta }\mathrm{b}}^{\ast 2}}$$
The whiteness and yellowness indices were also measured for standard CIE illuminant C and 2° field of view using the same software.