# Flavoromics Analysis of Passion Fruit-Roasted Chicken
## 4 Materials and Methods
### Determination of Primary Nutrients  
The content changes in the main components were studied during the 0-70 min roasting stage of passion fruit-roasted chicken. Amino acid analysis (Biochrom 30+, Biochrom Co., Ltd., Cambridge, UK) and ion chromatography (1260, Agilent, Santa Clara, CA, USA) were used to analyze the dynamic changes in flavor precursors such as free amino acids, free fatty acids, and soluble sugars in the samples during the roasting process. The infrared drying method (MA160-1 CN, Sedolis Scientific Instruments GmbH, Gilching, Germany) was used for the determination of moisture content, and the Kjeldahl nitrogen method (K9840, Shandong Haineng Scientific Instrument Co., Ltd., Jinan, China) was used for the determination of protein contents. Regarding free amino acids [51], 2.0 g of the chicken sample was added to a 50 mL volumetric flask and 20 mL of 5% sulfosalicylic acid was added, which was left to stand for 24 h. The supernatant (20 mL) was centrifuged for 15 min (6000 r/min) and was dried, adding 1 mL of sodium citrate buffer, and the amino acids were analyzed after membrane filtration (0.45 \(\upmu\)m). The UV detection wavelengths were 570 nm and 440 nm (Pro). By comparing the retention time and peak area of various amino acid standards, the types and contents of each amino acid were determined. Regarding nucleotides [52], 5.0 g of the passion fruit-roasted chicken meat sample was mixed with 20 mL of pre-cooled 5% perchloric acid and homogenized at 4 \({}^{\circ}\) C for 2 min (7000 r/min, 20 s, 6 times). After homogenization, a freeze centrifuge was used to remove protein (4000 r/min, 4 \({}^{\circ}\)C, 10 min). We then collected supernatant 1 and mixed the remaining residue with 10 mL of 5% perchloric acid again. Repeating the above process, supernatant 2 was collected, and supernatants 1 and 2 were combined. After adjusting pH to 6.0 with 5 mol/L KOH solution, it was quickly placed at 4 \({}^{\circ}\)C and stood for 1 h to precipitate potassium perchlorate and filtered (0.22 \(\upmu\)m) into an automatic injection bottle for testing by HPLC (1260, Agilent, USA). We then injected 5 \(\upmu\) L of sample and identified the nucleotide analyte from gradient elution at 254 nm. The column temperature was set to 30 \({}^{\circ}\)C, and mobile phase A was KH\({}_{2}\)PO\({}_{4}\) (0.02 mol/L, pH 7). Mobile phase B used high-performance liquid chromatography-grade methanol as the eluent. For separation, we adopted the gradient elution method with a constant flow rate of 1.0 mL/min and equilibrated the sample for 30 min before elution. The elution procedure was as follows: 0-3 min, 99% A; 3-3.5 min, 99% A-95% A; 3.5-5.5 min, 95% A; 5.5-6.5 min, 95% A-85% A; 6.5-9 min, 85% A; 9-10 min, 85% A-99% A. After washing, we rinsed the needle with a mixture of ultrapure water and methanol (1:1, \(v/v\)). For free fatty acids [53], 100 mg of passion fruit-roasted chicken was mixed well with 4 mL of the chloroform solution, centrifuged for 15 min (3500 r/min at a room temperature of 25 \({}^{\circ}\)C), stood for 1 h, and filtered. Adding 6 mL of the NaCl solution to the filtrate, the lower layer liquid 1 was transferred after centrifugation and the remaining liquid was mixed with 2 mL of dichloromethane. We then repeated the above operation, and the lower layer liquid 2 was aspirated. Liquids 1 and 2 were merged and dried with a nitrogen-purging instrument. GC-MS (6890A-5975C, Agilent, USA) was performed. The flow rate was 1.0 mL/min. The initial temperature was 100 \({}^{\circ}\)C, maintained for 5.0 min, and heated to 240 \({}^{\circ}\)C with a program of 4 \({}^{\circ}\)C/min for 15 min. The quality scanning range of MS was 30-550 \(m/z\).