# Upgrading of Mixed Food Industry Side-Streams by Solid-State Fermentation with _P. ostreatus_
## 2 Materials and Methods
### Assays  
Moisture, pH, ash, and total fat were determined, as previously described [9; 10], with minor modifications. In brief, moisture (%_w/w_) was determined by drying at 80 \({}^{\circ}\)C overnight. pH was determined after suspending 10 g of sample in 100 mL water. Ash (%_w/w_) was determined by burning accurately weighed (1-2 g) amounts of the samples in porcelain crucibles on a burner flame, followed by ashing at 550 \({}^{\circ}\)C for at least 3 h. Cheese whey was evaporated before burning. Total fat (%_w/w_) was determined by the extraction of 5 g of dried sample (105 \({}^{\circ}\)C; 3 h) with diethyl ether in a Soxhlet apparatus for about 2.5 h. The extract was condensed in a rotary evaporator, dried at 105 \({}^{\circ}\)C for 0.5 h, and weighed [9; 10]. In the case of cheese whey, the determination of fat was carried out according to the Gerber method for milk analysis, which involves the treatment with sulfuric acid and amyl alcohol for separation of fat, and measurement of the volume of fat after centrifugation. Fermentable sugars (sucrose, fructose, lactose, glucose, galactose) and ethanol, were analyzed by HPLC on a Shimadzu (Kyoto, Japan) chromatograph that was equipped with a SCR-101N stainless steel column, a LC-9A pump, a CTO-10A oven (set at 60 \({}^{\circ}\)C), and a RID-6A refractive index detector. Water was used as mobile phase (0.8 mL min\({}^{-1}\)) and a 1% \(v/v\) 1-butanol solution (Merck) was used as internal standard. Crude protein was analyzed according to the [PERSON]'s method for the determination of total nitrogen in milk [9]. The real protein increase (RPI) of the samples that was attributed to the microbial biomass was calculated using the formulas (Equations 1 and 2) [16]:  
\[RP=\frac{\left(M_{final}\times P_{final}\right)-\left(M_{initial}\times P_{ initial}\right)}{M_{initial}}+P_{initial} \tag{1}\]\[RPI=\frac{(\text{RP}-P_{initial})}{P_{initial}}\times 100 \tag{2}\]  
in which \(M_{initial}\) is the weight (g dry matter) before SSF, \(P_{initial}\) is the initial protein content (% dry matter), \(M_{final}\) is the weight (g dry matter) after SSF, and \(P_{final}\) is the final protein content (% dry matter). The determination of total phenolics in both organic (methanol) and the aqueous extracts of the samples was carried out spectrophotometrically, according to the Folin-Ciocalteau procedure [9; 17]. Gallic acid solutions in methanol were used as standards. In brief, methanolic extracts of the samples were prepared after alkaline hydrolysis with 1 N NaOH, followed by centrifugation, acidification with conc. HCl to pH 2, extraction with 1:1 diethyl ether/ethyl acetate, evaporation of the solvents, and finally, the dilution of the dry residue with 12 mL methanol. The total phenolics were determined by measuring the absorbance of the extract/Folin-Ciocalteau reagent mixture at 760 nm [17]. The determination of the antioxidant capacity in both organic (methanol) and the aqueous extracts was performed by the DPPH radical scavenging method, based on the decrease in absorbance at 517 nm of a methanolic solution of DPPH against aqueous methanol solution as blank [9; 17]. The determination of COD was carried out by both a colorimetric test [reaction with HgSO4, H2 SO4/AgSO4, and K2 Cr2O7, and titration of excess K2 Cr2O7 with FeSO4(NH4)2 SO4.6H2O and ferroin indicator], according to standard methods [9; 18], and a photometric test (using an electronic digestion device HACH DRB-200, a spectrophotometer HACH DR/2400, and digestion reagent vials HACH COD Digestion Vials, Low Range 3-150 mg/L COD) [9]. Metals Ca, Mg, Fe, and Cu were determined on a AA-6500 Series Atomic Absorption Spectrometer (Shimadzu) after ashing at 550 \({}^{\circ}\)C and dissolving in condensed nitric acid [10]. Major volatile compounds were determined by gas chromatography on a Shimadzu GC-8A system (Japan) with a stainless steel column TRB-624 (Teknokroma, Barcelona, Spain) (30 m, 0.32 mm i.d.