Organic acid and sugar content were evaluated using GC–MS (gas chromatography–mass spectrometry) following the procedures as described by Li et al. [29 (link)]. Soluble sugars and organic acids were extracted with 1.4 mL of 75% (V/V) methanol for 0.1 g of sample, with 400 ppm ribitol was used as internal standard, by shaking at 900 rpm at 70°C for 30 min. The supernatants were separated and transferred into a mixture of 750 μL chloroform (CHCl3) and 1.4 mL ddH2O. After mixing and centrifugation, samples of 2 μL and 50 μL of the supernatant were dried and then derivatized with 40 μL methoxyamine hydrochloride and 60 μL N-methyl-N-trimethylsilyl-trifluoroace-tamide (MSTFA). Analysis of soluble sugar and organic acid contents was conducted on a GCMS-2010SE instrument (Shimadzu Corporation, Kyoto, Japan).
The determination of starch content was performed as described by Li et al. [28 (link)]. The remaining precipitate after the extraction with 75% methanol for the determination of sugar and acid was repeatedly cleaned with 80% (v/v) ethanol three times, mixed with 0.1 M KOH and boiled for 30 min to gelatinize the starch. α-amylase at pH 4.5 was added and incubated at 55°C for 1 h. Reducing sugar content was determined by 3, 5-dinitrosalicylic acid (DNS), and then converted to starch content.
The determination of starch content was performed as described by Li et al. [28 (link)]. The remaining precipitate after the extraction with 75% methanol for the determination of sugar and acid was repeatedly cleaned with 80% (v/v) ethanol three times, mixed with 0.1 M KOH and boiled for 30 min to gelatinize the starch. α-amylase at pH 4.5 was added and incubated at 55°C for 1 h. Reducing sugar content was determined by 3, 5-dinitrosalicylic acid (DNS), and then converted to starch content.
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