Institute for Microbiology and Biotechnology, University of Bonn, 53115, Germany.
Institute for Microbiology and Biotechnology, University of Bonn, 53115, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 May 15;1172:122505. doi: 10.1016/j.jchromb.2020.122505. Epub 2020 Dec 19.
In recent decades, strategies to improve human health by modulating the gut microbiota have developed rapidly. One of the most prominent is the use of prebiotics, which can lead to a higher abundance of health-promoting microorganisms in the gut. Currently, oligosaccharides dominate the prebiotic sector due to their ability to promote the growth and activity of probiotic bacteria selectively. Extensive efforts are made to develop effective production strategies for the synthesis of prebiotic oligosaccharides, including the use of microbial enzymes. Within the genus Lactobacillus, several inulosucrases have been identified, which are suitable for the synthesis of prebiotic inulin-type fructooligosaccharides (inulin-FOS). In this study, a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 was used for the efficient synthesis of inulin-FOS. Product titers of 146.2 ± 7.4 g inulin-FOSL were achieved by the catalytic activity of the purified recombinant protein InuGB-V3. A time and resource-saving HPLC method for rapid analysis of inulin-FOS in isocratic mode was developed and optimized, allowing baseline separated analysis of inulin-FOS up to a degree of polymerization (DP) of five in less than six minutes. Long-chain inulin-FOS with a DP of 17 can be analyzed in under 45 min. The developed method offers the advantages of isocratic HPLC analysis, such as low flow rates, high sensitivity, and the use of a simple, inexpensive chromatographic setup. Furthermore, it provides high-resolution separation of long-chain inulin-FOS, which can usually only be achieved with gradient systems.
近几十年来，通过调节肠道微生物群来改善人类健康的策略发展迅速。其中最突出的是使用益生元，它可以导致肠道中促进健康的微生物数量增加。目前，由于能够有选择地促进益生菌的生长和活性，低聚糖在益生元领域占据主导地位。人们正在努力开发有效的生产策略来合成益生元低聚糖，包括使用微生物酶。在乳杆菌属中，已经鉴定出几种菊粉蔗糖酶，它们适合合成益生元菊粉型低聚果糖（菊粉-FOS）。在这项研究中，使用了短截版本的乳杆菌 DSM 20604 中的菊粉蔗糖酶来高效合成菊粉-FOS。通过纯化的重组蛋白 InuGB-V3 的催化活性，可获得 146.2±7.4g 菊粉-FOSL 的产物浓度。开发并优化了一种在等度模式下用于快速分析菊粉-FOS 的省时省力的 HPLC 方法，允许在不到六分钟的时间内分析聚合度（DP）高达五的菊粉-FOS 的基线分离分析。长链菊粉-FOS（DP 为 17）的分析时间可在 45 分钟以内完成。该方法具有等度 HPLC 分析的优点，例如低流速、高灵敏度以及使用简单、廉价的色谱设置。此外，它还提供了长链菊粉-FOS 的高分辨率分离，这通常只能通过梯度系统实现。
