Gas chromatography (GC) analysis
The fatty acid composition of FAMEs was analyzed on an Agilent gas chromatography unit (model 6890, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a DB-23 capillary column (30 m length, 0.25 μm film, 0.25 mm internal diameter; J&W Scientific, Agilent Technologies). The flow rate of the carrier gas (helium) was 5 mL/min and 30 mL/min at the make-up point; the injector and detector temperatures were 240 and 245 °C, respectively. The injection volume was 0.5 μL (splitless). The temperature program was as follows: initial temperature 60 °C, rate of 4 °C/min from 60 to 220 °C and hold for 10 min, and rate of 1 °C/min from 220 to 225 °C. Oven temperature program was set in order to obtain a good peak resolution. Identification of the FAME peaks was performed by comparing the retention times of the FAME standards; a model chromatogram is shown in Fig. 2. Agilent technologies software (G2072AA Rev.A.05.02 Chemstation) was used for integration of peaks. Fatty acid concentrations in mulberry fruits were expressed as percentage of total fatty acids and also were quantified unitarily in mg/100 g dw. Analyses were conducted in triplicate.