Free amino acid composition of ‘Asprinio_A’, ‘Asprinio_B’ and ‘Greco di Tufo’ wines were obtained by sampling 1 mL (in triplicate) per bottle. Subsequently, the samples were freeze-dried for amino acid extraction. The freeze-dried powder was subjected first to ethanol precipitation using 80% cold ethanol (1.0 mL), in the presence of nor-leucine (200 nmol) as internal standard, dissolved with a Teflon pestle, and centrifuged at 14,000× g at 4 °C. The supernatant was lyophilized, treated with 3% sulfosalicylic acid (500 μL) to precipitate any protein fraction still present, and recentrifuged [29 ,30 (link)]. Aliquots of samples (generally 30 μL) were directly analysed on a Biochrom-30 amino acid analyser (Biochrom, Cambridge, UK), equipped with a post-column ninhydrin derivatization system [31 ].
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