Departamento Bioactividad y Análisis de Alimentos, Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), CEI (UAM+CSIC), c/Nicolás Cabrera 9, 28049 Madrid, Spain.
J Agric Food Chem. 2012 Oct 24;60(42):10564-71. doi: 10.1021/jf303335m. Epub 2012 Oct 16.
This work describes an efficient enzymatic synthesis and NMR structural characterization of the trisaccharide β-D-galactopyranosyl-(1→4)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, also termed as lactulosucrose. This oligosaccharide was formed by the Leuconostoc mesenteroides B-512F dextransucrase-catalyzed transfer of the glucosyl residue from sucrose to the 2-hydroxyl group of the reducing unit of lactulose. The enzymatic reaction was carried out under optimal conditions, i.e., at 30 °C in 20 mM sodium acetate buffer with 0.34 mM CaCl(2) at pH 5.2, and the effect of factors such as reaction time (0-48 h), enzyme charge (0.8, 1.6, and 2.4 U mL(-1)), and sucrose:lactulose concentration ratios (20:40, 30:30, and 40:20, expressed in g/100 mL) on the formation of transfer products were studied. The highest formation in lactulosucrose was attained at 8 and 24-32 h by using 20%:40% and 30%:30% sucrose:lactulose mixtures, respectively, with 1.6 or 2.4 U mL(-1) dextransucrase, leading to lactulosucrose yields of 27-35% in weight respect to the initial amount of lactulose. Furthermore, minor tetra- and pentasaccharide, both probably derived from lactulose, were also detected and quantified. Likewise, the capacity of lactulosucrose to act as D-glucosyl donor once the sucrose was consumed, could explain its decrease from 16 to 24 h when the highest charge of dextransucrase was used. Considering the chemical structure of the synthesized oligosaccharides, lactulosucrose and its derivatives could potentially be excellent candidates for an emerging prebiotic ingredient.
本文描述了一种高效的酶促合成方法，并通过 NMR 结构表征，确定了三糖β-D-半乳糖基-(1→4)-β-D-果糖基-(2→1)-α-D-吡喃葡萄糖苷，也称为乳蔗糖的结构。该低聚糖由 Leuconostoc mesenteroides B-512F 葡聚糖蔗糖酶催化，从蔗糖转移葡萄糖基至乳果糖还原端的 2-羟基。在最佳条件下进行酶促反应，即在 30°C、20mM 乙酸钠缓冲液中，pH 值为 5.2，添加 0.34mM CaCl2，研究了反应时间（0-48 小时）、酶用量（0.8、1.6 和 2.4U·mL-1）、蔗糖：乳果糖浓度比（20:40、30:30 和 40:20，以 g/100mL 表示）等因素对转化产物形成的影响。使用 20%：40%和 30%：30%蔗糖：乳果糖混合物，分别在 1.6 或 2.4U·mL-1 葡聚糖蔗糖酶作用 8 小时和 24-32 小时，可获得最高的乳蔗糖生成量，转化率为 27-35%（相对于初始乳果糖量）。此外，还检测到并定量了少量四糖和五糖，它们可能均来源于乳果糖。同样，当使用最高浓度的葡聚糖蔗糖酶时，由于蔗糖的消耗，乳蔗糖作为 D-葡萄糖供体的能力从 16 小时下降至 24 小时，也可以解释这一现象。考虑到所合成低聚糖的化学结构，乳蔗糖及其衍生物可能是新兴益生元成分的极佳候选物。
