Enzymatic debittering of citrus juices: Optimization, modeling, and characterization of naringinase production from marine Bacillus subtilis strain BSnari

Abstract

The bitterness of citrus juices is a major obstacle to their industrial acceptance. Bitterness can be reduced using the naringinase enzyme. This study investigated two approaches for producing and purifying naringinase. Bacillus subtilis strain Bsnari was isolated from the Red Sea with accession number OQ576096 and had the highest naringinase productivity (7.09 ± 0.09 U/ml) using sucrose (1.5%) as carbon source, 0.6% w/v of citrus peel powder and 1% w/v of soy bean meal at pH 7.0 and 37 °C. Box-Behnken design has been successfully used for statistical modeling of naringinase production by Bacillus subtilis. Naringinase was partially purified using ammonium sulfate and gel filtration, resulting in a specific activity of 215.106 U/mg, a purification fold of 8.95, and a molecular weight of 73 KDa. Enzymatic hydrolysis reduced bitterness by around 33–36%, with optimal debittering after 4 h at 40–50 °C. A new method for processing citrus juice debittering was proposed using α-l-rhamnosidase and β-d-glucosidase encoded by the genes rhaA and bglA, respectively. The protein structures of β-l-rhamnosidase and β-d-glucosidase were also investigated. The topology of the 3D structure of α-l-rhamnosidase and β-d-glucosidase proteins of Bacillus sp. Predicted and aligned against other bacterial species. These findings provide insights into efficient naringinase production and purification for use in various applications, with implications for commercial production in food and pharmaceutical industries.