# Production of Starch Esters by Roasting Potato Starch with Unripe Apple Juice
## 2 Materials and Methods
### Production of Preparations of Potato Starch Roasted with Apple Juice Concentrate  
Native potato starch was saturated with the apple juice concentrate (having a concentration of 50\({}^{\circ}\)Bx) in the amount of 30 g dry matter per 100 g starch dry weight, conditioned at a temperature of 25 \({}^{\circ}\)C for 24 h, dried in an air-dryer (Mermert, Schwabach, Germany) at a temperature of 50 \({}^{\circ}\)C for 48 h, ground in a laboratory grinder, and sieved through a screen with a mesh size of 400 \(\upmu\)m. Native starch with the concentrate was roasted in an air-dryer (Mermert, Schwabach, Germany) at temperatures of 70, 80, 100, 120, or 140 \({}^{\circ}\)C for 3 h. The roasted preparations were rinsed with an aqueous solution of ethyl alcohol with a concentration of 65% until the supernatant became colorless (at an alcohol-to-preparation ratio of 3:1). The produced preparations were dried in an air-dryer (Mermert, Schwabach, Germany) at a temperature of 35 \({}^{\circ}\)C, ground, and sieved through a screen with a mesh size of 400 \(\upmu\)m. Preparations produced in an analogous way but without apple juice concentrate served as control samples. Qualitative and Quantitative Analysis of Organic Acids of the Unripe Apple Juice Concentrate by Means of a Liquid Chromatography-Mass Spectrometry Technique  
A sample of concentrated juice was 1000-fold diluted in water with purity suitable for mass spectrometry analyses. Concentrations of citric and malic acids were determined using an LCMS 8045 apparatus with an HPLC Prominence-i LC2030C 3D Plus unit (Shiamadzu, Kyoto, Japan). Analytes were separated on a Kinetex 2.6 \(\upmu\)m C18 100A 100 \(\times\) 3 mm column (Phenomenex, Torrance, CA, USA). Eluents used included a 0.1% solution of formic acid (A) and acetonitrile (B) and were applied in the following gradient system: start 10% B, 20% B in 5 min, 80% B in 10 min, 10% B in 13 min, and 10% B in 17 min. The acids were identified and quantified using the MRM negative ionization mode. In the case of citric acid, the precursor ion was selected at 191.4 \(m/z\) and the fragmentation ions at 110.95, 86.95, and 85.0, whereas in the case of malic acid, the precursor ion was selected at 149.4 \(m/z\) and the fragmentation ions at 86.90, 72.95, and 43.0. The acids were quantified against a 5-point calibration curve. Qualitative and Quantitative Analysis of Starch Esters by Means of High-Performance Liquid Chromatography (HPLC) Technique with the Calibration Curve Method  
Chromatographic analysis of organic acids was conducted after refining the preparations (which allows assuming that all free acids were removed from the sample) and alkaline deesterification of starch esters [17]. The preparation (20 g on dry matter basis) was transferred to a conical flask using 300 mL of distilled water. The suspension was brought to a boil and stirred until the preparation dissolved completely. After cooling, 1 L of rectified ethanol was poured into the flask to enable starch precipitation. After 24 h, the excess of a clear solution was decanted from above the precipitate. The refining process was repeated three times, and the resulting preparation was dried in an air-dryer (Mermert, Schwabach, Germany) at a temperature of 35 \({}^{\circ}\)C, ground, and sieved through a screen with a mesh size of 400 \(\upmu\)m. Next, alkaline deesterification was performed by mixing 2 g of the preparation (on a dry matter basis) and 100 mL of 0.5M NaOH on a magnetic stirrer at a temperature of 35 \({}^{\circ}\)C for 12 h. Afterward, 400 mL of rectified ethanol was added to a homogenous solution to enable starch precipitation. The obtained solution was concentrated by evaporating the solvent on a Rotavapor R II rotary evaporator (BUCHI, Flawil, Switzerland) at a compaction temperature of 55 \({}^{\circ}\)C and vacuum compaction of 120 mbar to a volume of 10 mL, and then 1 mL was filtered through a Millipore membrane filter with a pore size of 0.45 \(\upmu\)m and analyzed using high-performance liquid chromatography (HPLC).