1 University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Institute of Dairy Science and Probiotics, Groblje 3, 1230 Domžale, Slovenia.
Benef Microbes. 2015;6(4):573-81. doi: 10.3920/BM2014.0095. Epub 2015 Feb 12.
The aim of the study was to evaluate real-time PCR coupled with propidium monoazide (PMA) treatment for enumeration of microencapsulated probiotic lactobacilli microencapsulated in calcium alginate beads. Lactobacillus gasseri K7 (CCM 7710) and Lactobacillus delbrueckii subsp. bulgaricus (CCM 7712) were analysed by plate counting and PMA real-time PCR during storage at 4 °C for 90 days. PMA was effective in preventing PCR amplification of the target sequences of DNA released from heat-compromised bacteria. The values obtained by real-time PCR of non-treated samples were in general higher than those obtained by real-time PCR of PMA-treated samples or by plate counting, indicating the presence of sub-lethally injured cells. This study shows that plate count could not be completely replaced by culture independent method PMA real-time PCR for enumeration of probiotics, but may rather complement the well-established plate counting, providing useful information about the ratio of compromised bacteria in the samples.
本研究旨在评估实时 PCR 与吖啶橙（PMA）处理联用，以对包埋在海藻酸钙珠中的微囊化益生菌乳杆菌进行计数。在 4°C 下储存 90 天时，通过平板计数和 PMA 实时 PCR 对干酪乳杆菌 K7（CCM 7710）和德氏乳杆菌保加利亚亚种（CCM 7712）进行了分析。PMA 可有效防止热胁迫细菌释放的 DNA 目标序列的 PCR 扩增。未经处理样品的实时 PCR 获得的值通常高于经 PMA 处理样品或平板计数获得的值，表明存在亚致死损伤细胞。本研究表明，平板计数法不能完全被非培养依赖性方法 PMA 实时 PCR 替代，用于益生菌的计数，但可能会补充成熟的平板计数法，提供有关样品中受损细菌比例的有用信息。
