Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; Collaborative Drug Discovery Research (CDDR) Group, Pharmaceutical and Life Science Community of Research, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia.
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway 47500, Selangor Darul Ehsan, Malaysia; School of Pharmacy and Applied Science, La Trobe Institute of Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15;1092:145-151. doi: 10.1016/j.jchromb.2018.06.009. Epub 2018 Jun 5.
A quantitative assay using high-performance thin-layer chromatography (HPTLC) was developed to investigate bile salt hydrolase (BSH) activity in Pediococcus pentosaceus LAB6 and Lactobacillus plantarum LAB12 probiotic bacteria isolated from Malaysian fermented food. Lactic acid bacteria (LAB) were cultured in de Man Rogosa and Sharpe (MRS) broth containing 1 mmol/L of sodium-based glyco- and tauro-conjugated bile salts for 24 h. The cultures were centrifuged and the resultant cell free supernatant was subjected to chromatographic separation on a HPTLC plate. Conjugated bile salts were quantified by densitometric scans at 550 nm and results were compared to digital image analysis of chromatographic plates after derivatisation with anisaldehyde/sulfuric acid. Standard curves for bile salts determination with both methods show good linearity with high coefficient of determination (R) between 0.97 and 0.99. Method validation indicates good sensitivity with low relative standard deviation (RSD) (<10%), low limits of detection (LOD) of 0.4 versus 0.2 μg and limit of quantification (LOQ) of 1.4 versus 0.7 μg, for densitometric vs digital image analysis method, respectively. The bile salt hydrolase activity was found to be higher against glyco- than tauro-conjugated bile salts (LAB6; 100% vs >38%: LAB12; 100% vs >75%). The present findings strongly show that quantitative analysis via digitally-enhanced HPTLC offers a rapid quantitative analysis for deconjugation of bile salts by probiotics.
建立了一种使用高效薄层色谱（HPTLC）的定量分析方法，以研究从马来西亚发酵食品中分离的戊糖片球菌 LAB6 和植物乳杆菌 LAB12 益生菌中胆汁盐水解酶（BSH）的活性。将乳酸菌（LAB）在含有 1 mmol/L 基于钠的甘氨和牛磺结合胆盐的 de Man Rogosa 和 Sharpe（MRS）肉汤中培养 24 h。将培养物离心，所得无细胞上清液在 HPTLC 板上进行色谱分离。通过在 550nm 处的密度扫描定量结合胆盐，并将结果与衍生化后用茴香醛/硫酸的色谱板的数字图像分析进行比较。两种方法测定胆盐的标准曲线均具有良好的线性，相关系数（R）均在 0.97 至 0.99 之间。方法验证表明，两种方法均具有良好的灵敏度，相对标准偏差（RSD）均较低（<10%），检测限（LOD）分别为 0.4μg 和 0.2μg，定量限（LOQ）分别为 1.4μg 和 0.7μg。发现甘氨结合胆盐比牛磺结合胆盐的胆汁盐水解酶活性更高（LAB6；100%比>38%：LAB12；100%比>75%）。这些发现强烈表明，通过数字增强的 HPTLC 进行定量分析可为益生菌的胆汁盐去结合提供快速定量分析。
