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：天然發(fā)酵乳是微生物資源的豐富來源，其中一些具有益生菌特性，可幫助預(yù)防和治療人類疾病。然而，許多潛在的益生菌尚未采用傳統(tǒng)純培養(yǎng)方法進(jìn)行分離。培養(yǎng)組學(xué)，一種相對(duì)新穎的方法，主要用于培養(yǎng)和鑒定先前未知和未培養(yǎng)的細(xì)菌。盡管如此，其在天然發(fā)酵乳中的應(yīng)用仍然有限。因此，本研究的首要目標(biāo)是評(píng)估天然發(fā)酵乳中的細(xì)菌多樣性，并探索其微生物潛力。為此，我們從內(nèi)蒙古采集了三個(gè)天然發(fā)酵乳樣本，并在血培養(yǎng)瓶中進(jìn)行14天的富集培養(yǎng)。在0、3、7和14天，使用兩種培養(yǎng)溫度和四種培養(yǎng)基從樣本中分離出細(xì)菌，并對(duì)每個(gè)樣本在每個(gè)時(shí)間點(diǎn)進(jìn)行了全長16S rRNA基因測序。結(jié)果顯示，從三個(gè)樣本中分離出488個(gè)菌株，屬于3個(gè)門、11個(gè)屬和27個(gè)種。使用兩種培養(yǎng)溫度（20°C和30°C）和兩種培養(yǎng)基（MRS和RCM + Vb）在富集培養(yǎng)3、7和14天后，產(chǎn)生了大部分分離菌株。此外，全長16S rRNA基因測序證實(shí)，該方法顯著增加了天然發(fā)酵乳中的細(xì)菌多樣性?？傊囵B(yǎng)組學(xué)方法在分離更多細(xì)菌物種方面具有巨大潛力，有利于進(jìn)一步探索天然發(fā)酵乳中可培養(yǎng)微生物的多樣性。

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

關(guān)鍵詞：培養(yǎng)組學(xué)；富集培養(yǎng)；細(xì)菌多樣性；天然發(fā)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.

總結(jié)：該研究聚焦于利用培養(yǎng)組學(xué)（Culturomics）技術(shù)探索內(nèi)蒙古天然發(fā)酵乳中的細(xì)菌多樣性，以解決傳統(tǒng)純培養(yǎng)方法難以分離低豐度和未知菌株的問題。研究團(tuán)隊(duì)采集3份內(nèi)蒙古東部天然發(fā)酵乳樣本，通過血培養(yǎng)瓶富集培養(yǎng)14天，結(jié)合20℃和30℃兩種培養(yǎng)溫度、MRS、RCM+Vb等四種培養(yǎng)基進(jìn)行菌株分離，并采用PacBio全長16S rRNA基因測序技術(shù)鑒定。結(jié)果顯示，共分離出488株菌，分屬3門11屬27種，其中厚壁菌門占比最高（98.98%）；富集培養(yǎng)后樣本的OTU數(shù)量約為原始樣本的5倍，細(xì)菌門、屬、種數(shù)量分別增至原始樣本的8倍、5倍、3倍，且首次從天然發(fā)酵乳中分離出粗乳酸雙歧桿菌（Bifidobacterium crudilactis）、嘉義干酪乳桿菌（Lacticaseibacillus chiayiensis）等4種此前未被記錄的菌株。同時(shí)發(fā)現(xiàn)，20℃與 30℃組合培養(yǎng)、MRS和RCM+Vb培養(yǎng)基搭配，以及3天、7天、14天的富集培養(yǎng)周期組合，能顯著提升菌株分離效率，但14天富集后條件致病菌（如腸球菌屬、腸桿菌屬）占比升高。此外，臨床樣本分析表明，優(yōu)勢菌株如乳酸乳球菌（Lactococcus lactis）對(duì)發(fā)酵乳質(zhì)地和風(fēng)味有重要作用，部分新分離菌株可能具備益生菌潛力（如產(chǎn)短鏈脂肪酸、降膽固醇等）。該研究證實(shí)培養(yǎng)組學(xué)結(jié)合全長16S rRNA測序是探索天然發(fā)酵乳微生物多樣性的有效“自上而下”范式，為挖掘發(fā)酵乳中未知微生物及研究其與宿主相互作用提供了新依據(jù)。