PDF(Pubmed)
Abstract：
This study introduces an optimized integration of flow cytometry and fluorescence in situ hybridization (Flow-FISH) as an approach for the specific enumeration of gram-positive bacteria in probiotic products, overcoming the limitations of conventional methods. The enhanced Flow-FISH technique synergizes the rapid and automated capabilities of flow cytometry with the high specificity of FISH, facilitating the differentiation of viable cells at the species level within probiotic blends. By analyzing lyophilized samples of Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium animalis subsp. lactis, and a commercial product, the study highlights the optimized Flow-FISH protocol\'s advantages, including reduced hybridization times to 1.5 h and elimination of centrifugation steps. Comparative evaluations with the widely accepted enumeration methods plate count and Live/Dead (L/D) staining were conducted. The study revealed that Flow-FISH produces higher viable cell counts than plate count, thereby challenging the traditional \"gold standard\" by highlighting its predisposition to underestimate actual viable cell numbers. Against L/D staining, Flow-FISH achieved comparable results, which, despite the different foundational premises of each technique, confirms the accuracy and reliability of our method. In conclusion, the optimized Flow-FISH protocol represents a significant leap forward in probiotic research and quality control. This method provides a rapid, robust, and highly specific alternative for the enumeration of probiotic bacteria, surpassing traditional methodologies. Its ability to enable a more detailed and reliable analysis of probiotic products paves the way for precise quality control and research insights, underscoring its potential to improve the field significantly.
摘要：
这项研究引入了流式细胞术和荧光原位杂交（Flow-FISH）的优化整合，作为益生菌产品中革兰氏阳性细菌的特异性计数方法。克服了传统方法的局限性。增强的Flow-FISH技术将流式细胞术的快速和自动化能力与FISH的高特异性相结合，促进益生菌混合物中物种水平的活细胞分化。通过分析鼠李糖乳杆菌的冻干样品，植物乳杆菌，和动物双歧杆菌亚种。乳酸,和商业产品，该研究突出了优化的Flow-FISH协议的优势，包括减少杂交时间至1.5小时和消除离心步骤。用广泛接受的计数方法进行板计数和活/死(L/D)染色的比较评价。研究表明，Flow-FISH产生的活细胞计数高于平板计数，从而通过强调其低估实际可行细胞数量的倾向来挑战传统的“黄金标准”。针对L/D染色，Flow-FISH取得了可比的结果，which,尽管每种技术的基础前提不同，证实了我们方法的准确性和可靠性。总之,优化的Flow-FISH方案代表了益生菌研究和质量控制的重大飞跃。这种方法提供了一种快速、健壮,以及益生菌计数的高度特异性替代方法，超越传统方法。它能够对益生菌产品进行更详细和可靠的分析，为精确的质量控制和研究见解铺平了道路。强调其显著改善该领域的潜力。
