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Open Access
Fermentative Production of Debittered Kinnow Beverage Using a-L-Rhamnosidase Producing Yeast
(PAU, 2015) Singh, Pratiksha; Sahota, Param Pal
The production of -L- rhamnosidase from Clavispora lusitaniae (KF633446) has been characterized and evaluated for its effectiveness in debittering citrus juice. Clavispora lusitaniae produced 0.106 IU mL-1 enzyme activity in minimal media using rhamnose as the main carbon source. The enzyme has been purified to homogeneity by sulfate fractionation and DEAE- sephadex column chromatography, and measured by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be around 85 kDa. The enzyme has been purified to 10.1- fold with 46.98% recovery, 24.9 IU activity and 2.7 IU mg-1 specific activity. The pH and temperature optima of enzyme are 4 and 50 °C, respectively. The Michaelis- Menton constants for the hydrolysis of p-nitrophenyl -L-rhamnopyranoside are 0.18 mM and 25 IU mL-1. The enzyme is thermostable up to 50 °C and operational stability in kinnow juice. Metal ion K+ affected positively the activity and Ag+ completely inhibited the activity. The lyophilized enzyme powder could retain its enzymatic activity at 4 °C for 3 months. The enzyme (0.8 IU mL-1) in citrus juice could hydrolyze the naringin from 600 ppm to palatable bitterness (180.71 ppm). Oral acute toxicity study revealed the enzyme is nontoxic and safe for food use. The contents of arsenic (As), lead (Pb), cadmium (Cd) in the enzyme powder met the criteria for food use. The optimized parameters for debittering of kinnow juice are; enzyme activity (0.8 IU mL-1), TSS (13 °B), temperature (30±5°C) and incubation time (4 h) and for kinnow beverage; yeast inoculum concentration (0.75% v/v), TSS (13 °B), temperature (30±5 °C) and incubation time (48 h). These characteristics suggest that the -Lrhamnosidase from Clavispora lusitaniae holds potential for debittering the citrus juice, and the bioprocess consisting of production, salt precipitation, dialysis, ion exchange chromatography and lyophilization, a promising means to prepare the purified -Lrhamnosidase for commercially and setting up a strong base to enzymatically debittered citrus juice.
Open Access
Fermentative Production of Debittered Kinnow Beverage Using α-L-Rhamnosidase Producing Yeast
(Punjab Agricultural University, Ludhiana, 2016) Singh, Pratiksha; Sahota, Param Pal
The production of α-L- rhamnosidase from Clavispora lusitaniae (KF633446) has been characterized and evaluated for its effectiveness in debittering citrus juice. Clavispora lusitaniae produced 0.106 IU mL-1 enzyme activity in minimal media using rhamnose as the main carbon source. The enzyme has been purified to homogeneity by sulfate fractionation and DEAE- sephadex column chromatography, and measured by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be around 85 kDa. The enzyme has been purified to 10.1- fold with 46.98% recovery, 24.9 IU activity and 2.7 IU mg-1 specific activity. The pH and temperature optima of enzyme are 4 and 50 °C, respectively. The Michaelis- Menton constants for the hydrolysis of p-nitrophenyl α-L-rhamnopyranoside are 0.18 mM and 25 IU mL-1. The enzyme is thermostable up to 50 °C and operational stability in kinnow juice. Metal ion K+ affected positively the activity and Ag+ completely inhibited the activity. The lyophilized enzyme powder could retain its enzymatic activity at 4 °C for 3 months. The enzyme (0.8 IU mL-1) in citrus juice could hydrolyze the naringin from 600 ppm to palatable bitterness (180.71 ppm). Oral acute toxicity study revealed the enzyme is nontoxic and safe for food use. The contents of arsenic (As), lead (Pb), cadmium (Cd) in the enzyme powder met the criteria for food use. The optimized parameters for debittering of kinnow juice are; enzyme activity (0.8 IU mL-1), TSS (13 °B), temperature (30±5°C) and incubation time (4 h) and for kinnow beverage; yeast inoculum concentration (0.75% v/v), TSS (13 °B), temperature (30±5 °C) and incubation time (48 h). These characteristics suggest that the α-Lrhamnosidase from Clavispora lusitaniae holds potential for debittering the citrus juice, and the bioprocess consisting of production, salt precipitation, dialysis, ion exchange chromatography and lyophilization, a promising means to prepare the purified α-L- rhamnosidase for commercially and setting up a strong base to enzymatically debittered citrus juice.