Fermentative Production of Debittered Kinnow Beverage Using α-L-Rhamnosidase Producing Yeast
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Date
2016
Authors
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Publisher
Punjab Agricultural University, Ludhiana
Abstract
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.
Description
Keywords
Fermentative Production, kinnow juice