Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.
J Microbiol Methods. 2012 Jul;90(1):53-9. doi: 10.1016/j.mimet.2012.03.017. Epub 2012 Apr 5.
Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC). Oligosaccharide structures were confirmed by a combination of HILIC-HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3'-sialyllactose, disialyllactose, and 6'-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3'-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells.
许多研究已经证明了糖基化合物破坏微生物与黏膜上皮结合的能力。因此，低聚糖具有预防某些细菌性疾病的潜力。然而，目前用于鉴定抗黏附低聚糖的筛选方法存在局限性：它们既耗时又需要大量的低聚糖。需要开发分析技术，以便在使用感染的人细胞系模型之前，快速筛选和结构定义抗黏附低聚糖。有鉴于此，我们开发了一种快速筛选复杂低聚糖混合物对细菌潜在抗黏附活性的方法。我们的方法涉及使用全细菌细胞从溶液中“耗尽”游离低聚糖。作为一个案例研究，从荷斯坦弗里生奶牛的初乳中筛选游离低聚糖与整个大肠杆菌细胞的相互作用。通过高 pH 阴离子交换色谱和亲水相互作用液相色谱（HILIC-HPLC）测定低聚糖浓度的降低。通过 HILIC-HPLC、外切糖苷酶消化和离线负离子模式 MS/MS 的组合，确定低聚糖结构。耗尽测定法证实了某些牛低聚糖与细菌的选择性相互作用，先前的研究已经表明这些低聚糖与大肠杆菌相互作用。特别是，细菌细胞以群体依赖的方式耗尽以下低聚糖：3'-唾液乳糖、二唾液乳糖和 6'-唾液乳糖胺。该测定方法进一步通过研究得到验证，我们证明了 3'-唾液乳糖和牛初乳低聚糖混合物对大肠杆菌黏附分化的 HT-29 细胞的抑制活性。
