Oshiro Mugihito, Nakamura Keisuke, Tashiro Yukihiro
Laboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Biosci Biotechnol Biochem. 2025 Jan 24;89(2):294-303. doi: 10.1093/bbb/zbae173.
Lactic acid bacteria (LAB) shape diverse communities in fermented foods. Developing comprehensive quantification methods for community structure will revolutionize our understanding of food LAB microbiome. For this purpose, 16S rRNA gene amplicon-based quantification, using spiked exogenous bacterial cells as an internal standard, shows potential for comprehensiveness and accuracy. We validated cell spike-in amplicon sequencing for quantifying LAB communities in food. Low efficiency of LAB DNA extraction underscores the importance of compensating for DNA loss by spiking internal standard cells. Quantitative equations generated using 15 selected LAB mock species showed positive relationships between the ratio of MiSeq read counts and the expected 16S rRNA gene copy numbers, with coefficients of determination (R2) ≥ 0.6823. The fold differences between observed and expected 16S copy numbers were within the range of 1/3 to 3-fold. Our validation highlights that accurate preparation of the LAB mock community is crucial for cell spike-in amplicon sequencing accuracy.
乳酸菌（LAB）塑造了发酵食品中的多样群落。开发针对群落结构的全面定量方法将彻底改变我们对食品LAB微生物组的理解。为此，基于16S rRNA基因扩增子的定量方法，使用添加的外源细菌细胞作为内标，显示出全面性和准确性的潜力。我们验证了细胞加标扩增子测序用于定量食品中LAB群落的方法。LAB DNA提取效率低凸显了通过加标内标细胞来补偿DNA损失的重要性。使用15种选定的LAB模拟物种生成的定量方程显示，MiSeq读数计数比与预期的16S rRNA基因拷贝数之间呈正相关，决定系数（R2）≥0.6823。观察到的和预期的16S拷贝数之间的倍数差异在1/3至3倍范围内。我们的验证突出表明，准确制备LAB模拟群落对于细胞加标扩增子测序的准确性至关重要。
