The protein content was removed by precipitation (4 ◦C for 30 min) with trichloroacetic acid (final concentration 16 g/100 ml), followed by centrifugation (12,000 g, 15 min at 5 ◦C), filtration(0.22 m filters; Ministart high flow, Sartorius,
Goettingen,Germany) and injection (20 l) into an Aminex HPX-87 H column (300 mm × 7.8 mm, Bio Rad, USA). An isocratic mobile