Table_1_The power of DNA based methods in probiotic authentication.XLSX

IntroductionThe global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy.
MethodsA total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS).
ResultsTargeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5–V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3–V4 region data, ~95%–97% of total reads per sample corresponded to target species, while ~2%–3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product.
DiscussionWhile targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result.

## 引言
全球益生菌市场正处于快速增长阶段，为保障益生菌产品的功效与安全性，需实施严格的质量管控措施。益生菌产品的质量保障工作需涵盖：确认特定益生菌菌株的存在、测定活菌计数，以及验证样本中无污染菌株。业内建议益生菌生产商委托第三方开展益生菌质量及标签准确性评估。遵循该行业建议，本研究针对一款热销的多菌株益生菌产品的多批次样本，开展了标签准确性评估。

## 材料与方法
本研究共纳入55份样本（含5份多菌株成品与50份单菌株原料），涵盖共计100株益生菌，并采用联合分子检测方法开展分析，检测手段包括靶向PCR、非靶向扩增子高通量测序（High Throughput Sequencing, HTS）以及非靶向鸟枪宏基因组测序（Shotgun Metagenomic Sequencing, SMS）。

## 结果
采用物种特异性或菌株特异性PCR的靶向检测，成功验证了所有菌株/物种的身份。由于缺乏部分菌株的特异性鉴定方法，其中40株菌株可鉴定至菌株水平，剩余60株仅能鉴定至物种水平。
在扩增子HTS检测中，本研究靶向了16S rRNA基因的两个可变区。基于V5–V8可变区的测序数据，每份样本中约99%的总读段数比对至目标物种，未检出未声明物种。基于V3–V4可变区的测序数据，每份样本中约95%~97%的总读段数比对至目标物种，另有约2%~3%的读段数比对至未声明的变形杆菌属（Proteus）物种；但后续对变形杆菌的培养验证结果显示，所有批次样本均未检出活菌形式的变形杆菌。
鸟枪宏基因组测序得到的读段数，可在全部5份成品批次样本中比对组装至所有10株目标菌株的基因组。

## 讨论
尽管靶向检测方法可快速且精准地鉴定益生菌产品中的目标类群，但非靶向检测方法能够识别产品中所有物种（包括未声明物种），不过该类方法存在操作复杂、成本高昂、检测周期较长的局限性。