一株分离自婴儿肠道的乳双歧杆菌BL-99的菌株鉴定

Identification of Bifidobacterium animalis subsp.lactis BL-99 isolated from infant intestine

以婴儿肠道来源的乳双歧杆菌BL-99为研究对象,收集9株其他来源的动物双歧杆菌作为参考菌株,涵盖8株动物双歧杆菌乳亚种,1株动物双歧杆菌动物亚种。采用形态学、生理生化、基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)鉴定技术和全基因组测序技术,建立了适用于菌株BL-99的菌株鉴定方法。结果表明,菌株BL-99的MALDI-TOF MS鉴定比对值为2.38,与模式菌株的平均核苷酸一致性值为99.97%,鉴定为动物双歧杆菌(Bifidobacterium animalis),通过全基因组中groEL基因序列分析,进一步确定菌株BL-99为动物双歧杆菌乳亚种(Bifidobacterium animalis subsp.lactis)。对B.animalis subsp.lactis BL-99和参考菌株的1562个共有核心基因开展多位点序列分型分析,该菌株可与其他参考菌株有效区分。以该菌株的基因组序列为参考,开展单核苷酸多态性分析,不同培养代数的B.animalis subsp.lactis BL-99间的单核苷酸位点差异为0,各参考菌株与B.animalis subsp.lactis BL-99的单核苷酸位点差异均大于32,系统发育分析表明该方法可以有效区分B.animalis subsp.lactis BL-99与其他参考菌株。益生菌的安全性和功能性评价均在菌株水平具有特异性,研究建立的表型、基因型相结合的菌株鉴定方法可为菌株鉴定标准提供有效技术支撑,对促进益生菌在食品行业更加安全和广泛的应用具有重要意义。

Strain BL-99 isolated from the infant intestine was systematically identified using nine strains of Bifidobacterium animalis including B.animalis subsp.lactis and B.animalis subsp.animalis from different sources as reference.Microbial identification technologies including morphology,physiological and biochemical analysis,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)and whole genome sequencing were applied to establish the strain-level identification method suitable for the identification of strain BL-99.The MALDI-TOF MS identification score of strain BL-99 was 2.38,and the average nucleotide identity value of the strain BL-99 was 99.97%comparing with the type strain,which means the strain BL-99 was identified as Bifidobacterium animalis subsp.lactis based on colony morphology,physiological and biochemical properties,MALDI-TOF MS and whole genome sequencing using nine strains of B.animalis were the reference.Whole genome multilocus sequence typing analysis was carried out based on the 1562 core genes of B.animalis subsp.lactis BL-99 and other 9 reference strains,and the whole-genome multilocus sequence typing(wgMLST)results showed that strain BL-99 could be effectively distinguished from reference strains.Single nucleotide polymorphism(SNP)analysis showed that the difference in SNP between different generations of B.animalis subsp.lactis BL-99 was 0,and the reference strain was higher than 32.Phylogenetic analysis further demonstrated that the SNP analysis can effectively distinguish B.animalis subsp.lactis BL-99 from reference strains.The method that combined phenotype and genotype established could provide a technical basis for the establishment of strain identification relative standards and promote the safer and wider application of probiotics in the food industry.