| Summary: | Bifidobacteria are among the first colonizers of the human gastrointestinal tract. Different bacterial species use different mechanisms for utilization of various carbon sources in order to establish themselves in the complex microbial ecosystem of the gut. However, these mechanisms still need to be explored. Here, a large gene–phenotype correlation analysis was carried out to explore the metabolic and genetic diversity of bifidobacterial carbohydrate utilization abilities. In this study, we used 21 different carbohydrates to determine the growth phenotypes, the distribution of glycoside hydrolases (GHs), and gene clusters related to the utilization of multiple carbon sources in six human-residential <i>Bifidobacterium</i> species. Five carbohydrates significantly stimulated growth of almost all strains, while the remaining sugars exhibited species- and strain-specificity. Correspondingly, different <i>Bifidobacterium</i> species also had specific GHs involved in fermentation of plant or host glycans. Moreover, we analyzed several carbohydrate utilization gene clusters, such as 2-fucosyllactose (2′FL), sialic acid (SA), and fructooligosaccharide (FOS). In summary, by using 217 bifidobacterial strains and a wide range of growth substrates, our research revealed inter- and intra-species differences in bifidobacterial in terms of carbohydrate utilization. The findings of this study are useful for the process of developing prebiotics for optimum growth of probiotics, especially <i>Bifidobacterium</i> species.
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