## 2 Materials and Methods
### DSG Structural Composition Analysis
#### 2.3.3 Determination of Structural Carbohydrates and Lignin  
The lignin and carbohydrate content of extractive- and protein-free distiller's spent grain (DSG) was determined using the National Renewable Energy Laboratory (NREL) protocol for the determination of lignin and carbohydrates in biomass [36]. Briefly, 300 mg of extractive- and protein-free DSG was mixed with 3 mL of 72% sulfuric acid and kept for 1 h in a water bath preheated to 30 \({}^{\circ}\)C, with stirring every 5-10 min without removing the sample from the bath. After acid hydrolysis, the samples were removed from the water bath and diluted to a 4% concentration by adding 84 mL of deionized water. The mixture was then autoclaved at 121 \({}^{\circ}\)C for 1 h. After autoclaving, the samples were cooled and vacuum-filtered using pre-weighed and pre-dried filtering crucibles to separate the filtrate from the solid residue. The solid residue was dried in an oven at 105 \({}^{\circ}\)C until a constant weight was achieved, and this was measured as acid-insoluble residue (AIR).  
The crucible containing the acid-insoluble residue was placed in a muffle furnace equipped with a ramping program at 575 \({}^{\circ}\)C (SNOLT therm, Utena, Lithuania). After asking, the crucible was cooled, and the weight of the ash was recorded. The acid-insoluble lignin (AIL) was calculated by subtracting the weight of the ash from the weight of the acid-insoluble residue (AIR). Acid-soluble lignin (ASL) was determined by diluting 100 \(\upmu\)L of the filtrate with ultrapure water (1:20 dilution). The absorbance of the diluted filtrate was measured at a wavelength of 280 nm using a UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) [37]. The total lignin content on an extractive-free basis was calculated as the sum of acid-soluble lignin (ASL) and acid-insoluble lignin (AIL) using Equation (5).  
The carbohydrates in the filtrate were determined as cellulose (glucose content) and hemicellulose (the total sum of galactose, arabinose, and xylose) using Ultra High-Performance Liquid Chromatography (UHPLC, 1290, Agilent Technologies, Santa Clara, CA, USA) equipped with a DAD and RI detector set at 35 \({}^{\circ}\)C. Ten milliliters (10 mL) of the filtrate was neutralized with calcium carbonate, centrifuged, and filtered using a 0.2 \(\upmu\)m PTFE syringe filter (Sigma Aldrich, Darmstadt, Germany). The sugars were separated using a Pb-based carbohydrate analysis column (Aminex HPX-87P, BioRad, Richmond, CA, USA) at 80 \({}^{\circ}\)C with ultrapure water as the mobile phase at a flow rate of 0.6 mL/min. The concentration of each sugar was calculated from the area of the peaks using the equation of the line from the calibration curves of glucose, xylose, arabinose, and galactose standards. The percentage of sugars was calculated as the sum of glucose, xylose, arabinose, and galactose.  
\[\%\]  
Lignin (extractives and protein-free basis) = (% AIL + % ASL)  
where  
\[\%\]  
Extractives = percent extractives in the raw DSG sample.  
\[\%\]  
Protein = percent protein in the raw DSG sample.  
\[\%\]  
AIL = percent acid-insoluble lignin in the extractive- and protein-free DSG.  
\[\%\]  
ASL = percent acid-soluble lignin in the extractive- and protein-free DSG.