Culturomics: A promising approach for exploring bacterial diversity in natural fermented milk

Food Bioscience

Volume 62, December 2024, 105383

Yu Li, Xiaoqing Guo, Qingting Peng, Tingting Shen, Jianxia Yao, Yue Wei, Haiting Duan, Wenjun Liu

https://doi.org/10.1016/j.fbio.2024.105383

ABSTRACT：Natural fermented milk is an abundant source of microbial resources, some of which possess probiotic properties that can help prevent and treat human diseases. However, many potential probiotics have not yet been isolated using traditional pure-culture methods. Culturomics, a relatively novel approach, can be primarily employed to cultivate and identify previously unknown and uncultured bacteria. Nevertheless, their application in natural fermented milk remains limited. Therefore, the primary objective of this study was to evaluate the bacterial diversity in natural fermented milk and explore its microbial potential. For this purpose, we collected three natural fermented milk samples from Inner Mongolia and subjected them to enrichment culture in blood culture bottles for 14 d. Bacterial isolates were obtained from the samples at 0, 3, 7, and 14 days using two culture temperatures and four media, and full-length 16S rRNA gene sequencing was performed for each sample at each time point. The results revealed that 488 strains were isolated from three samples belonging to 3 phyla, 11 genera, and 27 species. The method using two culture temperatures (20 ?C and 30 ?C) and two media (MRS and RCM + Vb) yielded most of the isolates after 3, 7, and 14 days of enrichment culture. Furthermore, full-length 16S rRNA gene sequencing confirmed that this method significantly increased bacterial diversity in natural fermented milk. In conclusion, the culturomics method demonstrates great potential for the isolation of a greater number of bacterial species and is advantageous for further exploration of the diversity of culturable microorganisms in natural fermented milk.

摘要：天然发酵乳是微生物资源的丰富来源，其中一些具有益生菌特性，可帮助预防和治疗人类疾病。然而，许多潜在的益生菌尚未采用传统纯培养方法进行分离。培养组学，一种相对新颖的方法，主要用于培养和鉴定先前未知和未培养的细菌。尽管如此，其在天然发酵乳中的应用仍然有限。因此，本研究的首要目标是评估天然发酵乳中的细菌多样性，并探索其微生物潜力。为此，我们从内蒙古采集了三个天然发酵乳样本，并在血培养瓶中进行14天的富集培养。在0、3、7和14天，使用两种培养温度和四种培养基从样本中分离出细菌，并对每个样本在每个时间点进行了全长16S rRNA基因测序。结果显示，从三个样本中分离出488个菌株，属于3个门、11个属和27个种。使用两种培养温度（20°C和30°C）和两种培养基（MRS和RCM + Vb）在富集培养3、7和14天后，产生了大部分分离菌株。此外，全长16S rRNA基因测序证实，该方法显著增加了天然发酵乳中的细菌多样性。总之，培养组学方法在分离更多细菌物种方面具有巨大潜力，有利于进一步探索天然发酵乳中可培养微生物的多样性。

Keywords: Culturomics; Enrichment culture; Bacterial diversity; Natural fermented milk

关键词：培养组学；富集培养；细菌多样性；天然发酵乳

SAMMARY: This study focused on exploring bacterial diversity in naturally fermented milk from Inner Mongolia using culturomics, aiming to address the limitation of traditional pure-culture methods-their struggle to isolate low-abundance and previously unknown bacterial strains. The research team collected 3 samples of naturally fermented milk from eastern Inner Mongolia, subjected them to 14-day enrichment culture in blood culture bottles, isolated bacterial strains using two culture temperatures (20°C and 30°C) and four types of media (including MRS and RCM+Vb), and identified the strains via PacBio full-length 16S rRNA gene sequencing technology. Results showed that a total of 488 bacterial strains were isolated, belonging to 3 phyla, 11 genera, and 27 species, with Firmicutes accounting for the highest proportion (98.98%). After enrichment culture, the number of OTUs in the samples was approximately 5 times that of the original samples; the counts of bacterial phyla, genera, and species increased to 8 times, 5 times, and 3 times those of the original samples, respectively. Notably, 4 previously unrecorded bacterial strains-including Bifidobacterium crudilactis and Lacticaseibacillus chiayiensis-were isolated from naturally fermented milk for the first time. Meanwhile, the study found that three combinations significantly improved bacterial strain isolation efficiency: the combined use of 20°C and 30°C for culture, the pairing of MRS and RCM + Vb media, and the combination of 3-day, 7-day, and 14-day enrichment culture cycles. However, the proportion of conditional pathogenic bacteria (such as Enterococcus and Enterobacter) increased after 14 days of enrichment. In addition, analysis of clinical samples indicated that dominant strains such as Lactococcus lactis play a crucial role in shaping the texture and flavor of fermented milk, while some newly isolated strains may possess probiotic potential (such as in producing short-chain fatty acids and reducing cholesterol). This study confirmed that culturomics combined with full-length 16S rRNA sequencing constitutes an effective "top-down" paradigm for exploring the microbial diversity of naturally fermented milk, providing a new basis for mining unknown microbial resources in fermented milk and investigating their interactions with the host.

总结：该研究聚焦于利用培养组学（Culturomics）技术探索内蒙古天然发酵乳中的细菌多样性，以解决传统纯培养方法难以分离低丰度和未知菌株的问题。研究团队采集3份内蒙古东部天然发酵乳样本，通过血培养瓶富集培养14天，结合20℃和30℃两种培养温度、MRS、RCM+Vb等四种培养基进行菌株分离，并采用PacBio全长16S rRNA基因测序技术鉴定。结果显示，共分离出488株菌，分属3门11属27种，其中厚壁菌门占比最高（98.98%）；富集培养后样本的OTU数量约为原始样本的5倍，细菌门、属、种数量分别增至原始样本的8倍、5倍、3倍，且首次从天然发酵乳中分离出粗乳酸双歧杆菌（Bifidobacterium crudilactis）、嘉义干酪乳杆菌（Lacticaseibacillus chiayiensis）等4种此前未被记录的菌株。同时发现，20℃与 30℃组合培养、MRS和RCM+Vb培养基搭配，以及3天、7天、14天的富集培养周期组合，能显著提升菌株分离效率，但14天富集后条件致病菌（如肠球菌属、肠杆菌属）占比升高。此外，临床样本分析表明，优势菌株如乳酸乳球菌（Lactococcus lactis）对发酵乳质地和风味有重要作用，部分新分离菌株可能具备益生菌潜力（如产短链脂肪酸、降胆固醇等）。该研究证实培养组学结合全长16S rRNA测序是探索天然发酵乳微生物多样性的有效“自上而下”范式，为挖掘发酵乳中未知微生物及研究其与宿主相互作用提供了新依据。