Bioconversion of Cyanidin-3-rutinoside to Cyanidin-3-glucoside in Black Raspberry by Crude α-L-Rhamnosidase from Aspergillus Species

Abstract

The primary anthocyanins identified in black raspberry (Rubus occidentalis) were cyanidin-3-rutinoside (C3R), cyanidin-3-xylosylrutinoside (C3XR), and cyanidin-3-glucoside (C3G). C3G has been known to be more bioavailable than C3R, the most abundant anthocyanin in black raspberry. In this study, in order to enhance bioavailability of anthocyanins in black raspberry, bioconversion of C3R to C3G in black raspberry was conducted by cleaving terminal L-rhamnose of C3R using crude enzyme extracts (CEE) from A. usamii KCTC 6956, A. awamori KCCM 60380, A. niger KCCM 11724, A. oryzae KCCM 12698, and A. kawachii KCCM 32819. Each Aspergillus species was grown in a medium containing L-rhamnose as an inducer, and the supernatant of medium was concentrated by filtration, centrifugation, and ultrafiltration in every 24 h. The concentrated filter residue was dissolved in 50 mM sodium acetate buffer (pH 3.8) to obtain CEE. The α-L-rhamnosidase and β-D-glucosidase activities of the CEE were determined by a spectrophotometric method using p-nitrophenyl-rhamnopyranoside and p-nitrophenyl-glucopyranoside, respectively. Black raspberry juice (BRJ) (pH 3.8) was used as the substrate of bioconversion. The CEE from A. usamii cultured for 8 days had the highest α-L-rhamnosidase activity with 2.73 U/mL, followed by those from A. awamori, A. niger, A. oryzae, and A. kawachii. The CEE from A. usamii cultured for 8 days also had the highest β-D-glucosidase activity with 2.75 U/mL, followed by those from A. niger, A. awamori, A. kawachii, and A. oryzae. All the CEE from Aspergillus species, except for A. usamii, showed the highest α-L-rhamnosidase activity on the 7th day. α-L-Rhamnosidase had higher activity than β-D-glucosidase at all the times except for the CEE from A. usamii on the 8th day. The A. awamori CEE showed the highest α-L-rhamnosidase activity at 30 and 40 ˚C, while the enzyme activity of the A. usamii CEE was the highest at 50 and 60 ˚C. A. kawachii and A. oryzae had no significant enzyme activities under all tested conditions. When bioconversion of C3R to C3G in BRJ was analyzed by HPLC-DAD, the CEE from A. usamii and A. awamori hydrolyzed 95.7% and 95.6% of C3R to C3G, respectively, after 2 h incubation. The CEE from A. kawachii and A. oryzae did not convert C3R to C3G in BRJ. The result of the present study showed that A. usamii could be the most effective source for the bioconversion of C3R to C3G in BRJ.