The Submerged Culture Of Ganoderma Lucidum With Traditional Chinese Herbs And Its Tyrosinase Inhibitory Activity

Tyrosinase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis of mammalian. This study would provide a basement for the developing of whitening agents or tyrosinase-targeted medicine by screening new naturally occurring tyrosinase inhibitors. From 27 Traditional Chinese herbs Scutellaria baicalensis G. was successfully screened for the addition to the submerged culture of Ganoderma lucidum, and the fermentation conditions of the submerged culture of G. lucidum with herbs were optimized, the tyrosinase inhibitory activity of Ganoderma metabolites was studied. The main results of this dissertation were described as following:1.In vitro tyrosinase inhibitory activity was determined spectrophotometrically by measuring the accumulation of o-diquinone at 420nm using catechol as substrate. The optimum reaction pH and temperature of potato tyrosinase were 6.5 and 30°C respectively. The tyrosinase inhibitory activity of 27 Traditional Chinese herbs extracted with water and fermented with G. lucidum was investigated according to the established tyrosinase-detecting system. Result shows that the tyrsinase inhibitory activity of 6 g/L Scutellaria baicalensisi, Myrica rubra, Camellia sinensis, Aloe vera, Moraceae alba and Ginkgo biloba fermented with G. lucidum respectively increased by 31.80%, 21.2%, 17.94%, 17.57%, 25.83% and 14.29% compared with the tyrosinase inhibitory activity of the native herbs extracted with water.2.Orthogonal design L16(45) and response surface methodology were successfully applied to optimize the medium composition which affects the tyrosinase inhibitors production by Ganoderma lucidum. The optimum medium composition was determined as 6.3 g/L of S. baicalensis, 12.0 g/L of bran meal, 6.0 g/L of peptone, 15.6 g/L of corn flour, 6.8 g/L of glucose, 11.3 g/L of soy meal and 1.5 g/L of KH2PO4. The fermentation conditions which affect the tyrosinase inhibitors production by the submerged culture of G. lucidum were studied. The tyrosinase inhibitory activity reaches its maximum at temperature of 30°C and shaking speed of 150 rpm with 150 mL loading volume in a 500 mL flask. Tyrosinase inhibitory activity (TIA) of the submerged culture reaches its maximum value 53.47% at a raw inhibitor (the lyophilized powder of fermented broth) concentration of 3.9 mg/mL, which is accordant to the predicted value 52.87% under the optimized fermentation conditions. Confirmatory experiments performed in a 25.0 L reactor showed that the maximum TIA of the submerged culture was 59.47% at 120 h.3.Effects of S. baicalensis on the submerged culture of G. lucidum were studied under the optimized conditions. The pH shift of submerged culture was much slower and the tyrosinase inhibitory activity of submerged culture was much higher with the addition of S. baicalensis in the culture. The mechanism of S. baicalensis transformed by G. lucidum was investigated using rutin as substrate. Our investigation shows that theβ-glucosidase of G. lucidum could exert a positive effect on the process of rutin transformed into quercetin-3-O-β-D-glucoside. The space hindrance of flavone interaction with tyrosinase was...