# Consumer Characterization of Wet- and Dry-Aged Mutton Flavor Profile Using Check-All-That-Apply
## 2 Materials and Methods
### Chemical Analysis
#### 2.2.2 Fatty Acid Methyl Ester (FAME)  
Muscle fatty acids contribute to the generation of several important odor active volatile compounds during the storage and cooking of meat [47], and the effect of dry-ageing on mutton fatty acids is unknown. Therefore, the FAME profile for the raw wet- and dry-aged patties was determined using the methodology outlined below.  
For both treatments (wet- and dry-ageing), raw patty mince was analyzed for FAME using the methods described in [PERSON] et al. [48] and [PERSON] et al. [49]. All reagents and chromatography supplies were purchased from Sigma-Aldrich, Macquarie Park, New South Wales, Australia. Chemicals used in the analysis included internal standard (nonadecanoic acid methyl ester (C19:0) part no. 74208-1G), reagent-grade potassium hydroxide (part no. 1050331000), HPLC-grade methanol (part no. MA004-2.5L-J), AR-grade sulfuric acid (part no. 258105-2.5L-PC), reagent-grade hexane (RP1083-G2.5L), external standard fatty acid methyl ester (FAME) reference standard C8-C24 mix (part no. CRM18918).  
Duplicate 1.0 g aliquots of frozen ground raw sample for each treatment (wet- and dry-aged) were weighed into 10 mL test tubes, 1 mL of internal standard (0.5 mg/mL nonadecanoic acid methyl ester in methanol) was added, followed by 0.7 mL of 10% KOH in water and 5.3 mL of methanol. Tubes were then mixed on a vortex mixer. The tubes were incubated at 55 \({}^{\circ}\)C for 1.5 h and shaken vigorously every 20 min. They were then cooled to room temperature under running water and then 0.6 mL of 24 N sulfuric acid in water was added and tube contents mixed. The incubation, shaking and cooling steps were repeated, then 3 mL of hexane was added to the tube and the contents were mixed for 5 min. Tubes were then centrifuged at 2000 rpm for 10 min with 1 mL of hexane layer collected into a 2 mL GC vial ready for analysis.  
GC analysis was conducted on an Agilent 7890B GC system fitted with an Agilent 7693 autosampler, a flame ionization (FID) detector and a 25 m SGE-70 capillary column, internal diameter of 0.32 mm, 0.5 \(\upmu\)m film thickness (part number 054606, Trajan Scientific and Medical, Ringwood, Australia). Helium was used as the carrier gas at a flow rate of 0.3 mL/min and the FID was supplied with 30 mL/min hydrogen, 300 mL/min air and nitrogen make-up gas at 30 mL/min. The GC was programmed for split injection (30:1) with the injector maintained at 260 \({}^{\circ}\)C throughout, and the FID at 260 \({}^{\circ}\)C. The oven program was initiated at 140 \({}^{\circ}\)C, where it was held for 5 min, then increased at 4 \({}^{\circ}\)C/min until it reached 240 \({}^{\circ}\)C, where it was held for 20 min. An injection volume of 1 \(\upmu\)L was used for external standard and sample analysis.  
Identification of fatty acids was based on retention time which was matched with the external FAME standard. Quantification of the sample FAMEs was conducted using FAME external standard curves over a concentration range 0.1 mg/g to 1.02 mg/g. All standard curves had an R\({}^{2}\) value > 0.997; FAME results are expressed as FAME mg/g meat.