## 2 Materials and Methods
### Cytotoxicity Assessment by MTT and LDH  
Evaluation of cytotoxicity requires sensitive, quantitative, reliable and automated methods for the precise determination of cell viability and death, therefore, we selected the MTT and LDH assays for this task.  
The mouse fibroblasts L929 cell line (ECACC--European Collection of Authenticated Cell Cultures) was selected for cytotoxicity assessment of pea powder, almond powder, yeast powder, spirulina powder and _Pleutrotus_ spp. flour. All samples were analyzed from a starting concentration of 500 mg/mL and serially diluted in order to obtain the following concentrations: 250 mg/mL, 62.5 mg/mL, 7.813 mg/mL, and 0.651 mg/mL.  
Cultivation of L929 cells was performed according to ECACC specifications in DMEM (Dulbecco's Modified Eagle Medium, Sigma-Aldrich, St. Louis, MO, USA) media supplemented with 10% FBS (Fetal Bovine Serum--Sigma-Aldrich) and 1% Pen/Strep (penicillin/streptomycin solution, 50 ug/mL--Sigma-Aldrich) for 24 h at 37 \({}^{\circ}\)C, at 95% humidity with 5% CO\({}_{2}\). After 24 h cells were washed using PBS (Phosphate Buffered Solution--Sigma-Aldrich), harvested using trypsin (Sigma-Aldrich) and counted using Trypan Blue (Sigma-Aldrich) and a hemocytometer. The seeding density for the MTT and LDH assays was optimized at 5 \(\times\) 10\({}^{4}\).  
Using the MTT we measured the cellular metabolic activity, which is an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide or MTT) to purple formazan crystals by metabolically active cells [55]. The mechanism of function relies on viable cells containing NAD(P)H-dependent oxidoreductase enzymes that reduce the MTT to formazan. Moreover, the insoluble formazan crystals formed are dissolved using a cell grade solvent and the resulting colored solution is quantified by measuring its absorbance at 500-600 nm, depending on various kits specifications. This non-radioactive, colorimetric assay system using MTT was first described by [PERSON]. (1983) [56] and improved in subsequent years by several other investigators. Since disturbances in cellular metabolism cannot fully confirm the actual toxicity of a certain compound, elucidation of the process associated with reduction of cellular population is important, therefore we selected LDH assay for quantification of plasma membrane damage. Upon cellular damage, NAD\({}^{+}\) is reduced to NADH/H\({}^{+}\) by the LDH-catalyzed conversion of lactate to pyruvate. Subsequently, the catalyst transfers H/H+ from NADH/H+ to the tetrazolium salt INT which is reduced to formazan. An increase in the amount of dead or plasma membrane-damaged cells results in an increase in LDH enzyme activity in the culture supernatant [56].  
Cells seeded at 5 \(\times\) 10\({}^{4}\) density in a clear 96 well cell culture plate were treated with pea powder, almond powder, yeast powder, spirulina powder and _Pleurotus_ spp. flour (samples concentrations: 500 mg/mL, 250 mg/mL, 62.5 mg/mL, 7.813 mg/mL, and 0.651 mg/mL) and incubated for 24 h at 37 \({}^{\circ}\)C, 95% humidity with 5% CO\({}_{2}\). After 24 h of exposure to tested compounds, cells were incubated for 4 h with MTT reagent (Roche) at 37 \({}^{\circ}\)C, 95% humidity with 5% CO\({}_{2}\). After incubation, cells were treated with MTT solvent (Roche) for 15 min at room temperature. Absorbance was measured using a spectrophotometric microplate reader (ELISA reader) at OD = 570 nm. For the LDH assay we used the LDH Cytotoxicity Detection Kit (Roche). Cells exposed to tested compounds were treated with LDH reagent mixture for 15 min at 37 \({}^{\circ}\)C. Afterwards, the LDH activity was measured using a spectrophotometric microplate reader (ELISA reader) at 492 nm with a 600 nm wavelength reference.