Benucci Ilaria, Graziano Monica Mollica, Lombardelli Claudio, Oliveri Paola, Vinciguerra Vittorio, Esti Marco
Department of Agriculture and Forest Sciences (DAFNE), Tuscia University, via S. Camillo de Lellis snc, 01100 Viterbo, Italy.
Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF), Tuscia University, Viterbo 01100, Italy.
Food Res Int. 2025 Nov;219:117163. doi: 10.1016/j.foodres.2025.117163. Epub 2025 Jul 29.
The demand for natural sweeteners as alternatives to sucrose is growing rapidly, driving research into enzymatic bioconversion methods for more efficient production. Glycyrrhizin (GL) is approximately 190 times sweeter than sucrose, but its excessive consumption has been linked to adverse health effects. Its hydrolysis yields glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG), a compound nearly 1000 times sweeter than sucrose and with improved sensory and solubility properties. However, the selective conversion of GL into GAMG remains a challenge due to the simultaneous formation of glycyrrhetinic acid (GA) as a byproduct. This study aimed to characterize β-D-glucuronidase (β-GUS) from three different sources (bovine liver, recombinant Escherichia coli, and limpets, Patella vulgata) to identify the most efficient biocatalyst for the selective hydrolysis of GL. β-D-Glucuronidases were characterized for optimal pH, temperature, and catalytic properties using the synthetic substrate 4-nitrophenyl-β-D-glucuronide (pNPG). The enzymes were tested for bioconversion of GL. Among them, β-GUS from Patella vulgata showed the highest efficiency, reducing GL content by 82 % in 72 h, producing 57 mg/L of GAMG and 46 mg/L of GA in 6 h at 40 °C. β-GUS was immobilized on chitosan beads, enhancing specific activity and storage stability (retaining 70 % of initial activity after 30 days). Maximum GAMG production was 61 mg/L in 48 h at 40 °C, with a 58 % GL reduction, and 40 mg/L in 6 h at 80 °C, with a 45 % GL reduction. Irrespective of the treatment temperature, the immobilized biocatalyst allowed the production of the intensive sweetener in a nearly pure form.
作为蔗糖替代品的天然甜味剂需求正在迅速增长，这推动了对更高效生产的酶促生物转化方法的研究。甘草甜素（GL）的甜度约为蔗糖的190倍，但其过量食用与不良健康影响有关。其水解产生甘草次酸3 - O - 单 - β - D - 葡萄糖醛酸苷（GAMG），该化合物的甜度比蔗糖高近1000倍，且具有改善的感官和溶解性。然而，由于同时形成副产物甘草次酸（GA），将GL选择性转化为GAMG仍然是一个挑战。本研究旨在表征来自三种不同来源（牛肝、重组大肠杆菌和帽贝，即普通帽贝）的β - D - 葡萄糖醛酸酶（β - GUS），以确定用于GL选择性水解的最有效生物催化剂。使用合成底物4 - 硝基苯基 - β - D - 葡萄糖醛酸苷（pNPG）对β - D - 葡萄糖醛酸酶的最佳pH、温度和催化特性进行了表征。测试了这些酶对GL的生物转化。其中，来自普通帽贝的β - GUS效率最高，在72小时内使GL含量降低了82%，在40°C下6小时内产生了57mg/L的GAMG和46mg/L的GA。β - GUS固定在壳聚糖珠上，提高了比活性和储存稳定性（30天后保留70%的初始活性）。在40°C下48小时内最大GAMG产量为61mg/L，GL降低58%，在80°C下6小时内为40mg/L，GL降低45%。无论处理温度如何，固定化生物催化剂都能以几乎纯的形式生产这种高强度甜味剂。
