# Impact of Ageing on Pea Protein Volatile Compounds and Correlation with Odor
## 3 Materials and Methods
### Volatile Compounds Analysis  
The volatile compounds extraction was done using headspace solid micro-extraction and the analysis using gas chromatography coupled with mass spectrophotometry (HS-SPME-GC-MS). For each treatment at each sampling time, volatile compounds analysis was run in triplicates, using a previously optimized method for PPI [35].  
From the 2 g aliquot, a 0.2 g PPI sample was weighted directly in a new clear 20 mL extraction vial (VA201) capped with septum caps (18 mm caps, 8 mm PTFE/silicon septum, SACA001), all purchased from JASCO, France. Distilled water was added to obtain a 2 mL suspension at 10% (\(w/v\)) and a liquid/gas ratio of 2/18 (\(v/v\)). A SPME device containing a 1 cm fused-silica fiber coated with a 50/30 \(\upmu\)m thickness of DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) was used for HS-SPME extraction. This fiber was selected to ensure the best extraction of a diversity of volatile compounds [9; 10; 36; 37; 38]. The fiber (24 Ga 50/30 \(\upmu\)m, for manual holder, 3 pK, 57328-U) was purchased from Sigma and used with a manual fiber holder. The extractions were carried out in an electro thermal magnetic stirrer with a water bath (MS-H-Pro+, DLAB) to ensure a homogeneous temperature and constant agitation for the sample and headspace. The fiber was conditioned before analysis by heating it in the gas chromatograph injection port at 270 \({}^{\circ}\)C for 30 min, according to the manufacturer's specifications. Equilibrium step and extraction step were conducted both at 40 \({}^{\circ}\)C with agitation at 350 rpm in the dark. The equilibrium time was30 min and the extraction time, exposure of the fiber in the headspace of the vial was 60 min [9; 10; 13; 36; 37; 38; 39; 40; 41; 42; 43].  
An HP 6890 Series Gas Chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with an HP 5973 Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA) (Quadrupole) was used with a DB-WAX column (30 m \(\times\) 0.32 mm \(\times\) 0.25 um, 123-7032, Agilent, J&W Scientific, Santa Clara, United States) to analyze the compounds of interest [10; 13; 21; 23]. The SPME fiber was desorbed and maintained in the injection port at 250 \({}^{\circ}\)C for 5 min. The sample was injected in split mode, with a purge flow of 140 mL/min at 0 min to generate sharp, well-separated peaks on the chromatograph. Helium was used as a carrier gas at 1.4 mL/min with a linear velocity of 43 cm/s. The programmed temperature, selected from preliminary trials, was isothermal at 40 \({}^{\circ}\)C for 3 min, raised to 100 \({}^{\circ}\)C at a rate of 3 \({}^{\circ}\)C/min, and then raised to 230 \({}^{\circ}\)C at a rate of 5 \({}^{\circ}\)C/min and held for 10 min. The total run time was 59 min [13; 21]. The ionization source and transfer line temperatures were set respectively at 230 \({}^{\circ}\)C and 190 \({}^{\circ}\)C.  
The mass spectra were obtained using a mass selective detector with an electron impact voltage of 70 eV in full scan mode over the range \(m/z\) 29 to 400. Compounds were identified by comparing their mass spectra with NIST 08 (National Institute of Standards and Technology), Wiley, and INRA libraries, with a low integration limit of 50,000 in peak area, allowing the best peak identification.