# The Effect of Type and Duration of Digestive Enzyme Treatment on Coffee Bean Composition
## 2 Materials and Methods
### Chemical Composition of Coffee  
Analyses of coffee chemical composition were divided into two groups: basic components (carbohydrates and protein contents) and bioactive compounds that are characteristic in coffee (caffeine, total polyphenols, 3-CQA).  
The content of coffee carbohydrates (fructose, glucose, sucrose) was analyzed with the HPLC system (Shimadzu Europa GmbH, Duisburg, Germany) equipped with an evaporative light-scattering detector [15]. Methanolic extracts were diluted and filtered using syringe filters (diameter 25 mm, pore size 0.22 \(\upmu\)m). Carbohydrate separation was performed on the Suplecoil LC-NH2 column (25 cm \(\times\) 4.6 mm, 5 \(\upmu\)m) at 30 \({}^{\circ}\)C. Injection volume was 5/10 \(\upmu\)L for sucrose and 10-25 \(\upmu\)L for fructose and glucose analyses. The mobile phase was acetonitrile and water (78:22), at a flow rate of 1 mL/min. Total time of analysis was 12 min. Determination of carbohydrates was based on comparison with retention times observed for standard solutions (2 mg/mL): fructose--6.767 min, glucose--7.525 min and sucrose--10.108 min. Calibration curves for each analyzed sugar were prepared in the LabSolution program and used for further calculations. Limit of detection (LOD) and limit of quantification (LOQ) were as follows: fructose--LOD 0.160 \(\upmu\)g, LOQ 0.490 \(\upmu\)g; glucose--LOD 0.204 \(\upmu\)g, LOQ 0.617 \(\upmu\)g; sucrose--LOD 0.130 \(\upmu\)g, LOQ 0.380 \(\upmu\)g.  
Protein content was evaluated using the Lowry method [16]. The reagent mixture was prepared by combining the following solutions in 10:1:1 ratio: 2% Na2 CO3 in 0.1 M NaOH, 1% CuSO4 and 2% KNaC4H4O6.4H2O. Diluted SDS extracts (0.6 mL) were mixed with 3 mL of reagent mixture. After 10 min, 0.3 mL of Folin-Ciocalteu reagent diluted with water (1:1) was added. Samples were placed in the dark for 30 min. Absorbance was measured at 750 nm (UVmini 1240, Shimadzu, Kioto, Japan) against a reagent blank. A standard solution of bovine serum albumin in water (1.5 mg/mL) was used to prepare a calibration curve, which was used in further calculations.  
Total polyphenols were analyzed with the Folin-Ciocalteu method described by [PERSON] et al. [17]. GA solution in 80% methanol (1 mg/mL) was used as a standard solution to prepare the calibration curve.  
The amounts of caffeine and 3-CQA were determined using an HPLC system with a diode array detector, using a method described by [PERSON] et al. [18] with modifications. Samples were prepared as in carbohydrate analysis. Separation was performed on theKinetex 5u C18 100A column (150 \(\times\) 4.6 mm) at 30 \({}^{\circ}\)C, with injection volume of 10 \(\upmu\)L, mobile phase of acetic acid solution (0.1%) and acetonitrile mixed in an 87:12 ratio, flow rate of 1 mL/min and for a total time of 12 min. Caffeine was analyzed at 272 nm, and 3-CQA was measured at 325 nm. Determination of compounds was based on comparison of retention times with values established for standard solutions (2 mg/mL): 3.552 min and 3.072 min for caffeine and 3-CQA, respectively. Standard solutions were used for calibration curves preparation and further calculations in LabSolutions software. LOD and LOQ for caffeine were 0.005 \(\upmu\)g and 0.151 \(\upmu\)g, respectively, and for 3-CQA were 0.005 \(\upmu\)g and 0.145 \(\upmu\)g, respectively.