Identification of Bifidobacterium animalis subsp. lactis BL-99 isolated from infant intestine
YU Xuejian1, LIU Jinfeng2,3, ZHAO Ting1, MA Xia2,3, LIU Yiru1, LIU Weixian2,3, CHENG Kun1, ZHANG Xin1, CAO Yanhua1, XIN Di1, FENG Huijun1, LIU Fudong2,3, ZHAO Wen2,3, HONG Weilian2,3, YAO Su1*
1(China National Research Institute of Food and Fermentation Industries Co. Ltd., China Center of Industrial Culture Collection, Beijing 100015, China) 2(Inner Mongolia Dairy Technology Research Institute Co. Ltd., Hohhot 010110, China) 3(Inner Mongolia Yili Industrial Group Co. Ltd., Hohhot 010110, China)
Abstract: 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 1 562 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.
 FAO/WHO.Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food[R].London,Ontario,Canada,April 30 and May 1,2002.  中国卫生部. 可用于食品的菌种名单[Z].2010,65号. Ministry of Health,P.R.China,List of strains that can be used for food[Z].2010,No.65.  IPA.Guidelines to qualify a microorganism to be termed as “probiotic”.International Probiotics Association (IPA)[Z].2017.1  中国食品科学技术学会益生菌分会. 益生菌的科学共识(2020年版)[J].中国食品学报,2020,20(5):303-307. Probiotics Society of the Chinese Institute of Food Science and Technology,Scientific consensus on probiotics (2020)[J].Journal of Chinese Institute of Food Science and Technology,2020,20(5):303-307.  洪维鍊, 刘伟贤,赵雯,等.一种可调节胃肠道菌群的乳双歧杆菌BL-99及其应用:中国,CN110964655A[P].2020-04-07. HONG W L,LIU W X,ZHAO W,et al.A Bifidobacterium lactis BL-99 that can regulate gastrointestinal flora and uses thereof:China,CN110964655A[P].2020-04-07.  洪维鍊, 刘伟贤,赵雯,等.一种可提高免疫力的乳双歧杆菌BL-99及其应用:中国,CN110964657A[P].2020-04-07. HONG W L,LIU W X,ZHAO W,et al.A Bifidobacterium lactis BL-99 that can enhance immunity and uses thereof:China,CN110964657A[P].2020-04-07.  刘伟贤, 洪维鍊,赵雯,等.乳双歧杆菌BL-99的新应用:中国,CN110893193A[P].2020-03-20. LIU W X,HONG W L,ZHAO W,et al.New use of Bifidobacterium lactis BL-99:China,CN110893193A[P].2020-03-20.  赵雯, 刘伟贤,洪维鍊,等.乳双歧杆菌BL-99在抑制肠道炎症方面的新应用:中国,CN110893194A[P],2020-03-20. ZHAO W,LIU W X,HONG W L,et al.New use of Bifidobacterium lactis BL-99 in inhibiting intestinal inflammation:China,CN110893194A[P].2020-03-20.  HELLER K J,BOCKELMANN W,BROCKMANN E,et al.Identification of probiotics at strain level:Guidance document[J].Bulletin of the International Dairy Federation,2013,B462:4-5.  国家农业标准化检测与研究中心, 中国测试技术研究院,深圳出入境检验检疫局,等.GB/T 33682—2017 基质辅助激光解析电离飞行时间质谱鉴别微生物方法通则[S].Beijing:中国标准出版社,2017. National Agricultural Standardization Testing and Research Center,China Institute of Testing Technology,Shenzhen Entry-Exit Inspection and Quarantine Bureau,et al.GB/T 33682—2017 General microganism identification method with matrix-assisted laser desorption/ionization time of flight mass spectrometry[S].Beijing:China Standards Press,2017.  KIM M,OH H S,PARK S C,et al.Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes[J].International Journal of Systematic and Evolutionary Microbiology,2014,64(Pt 2):346-351.  GORIS J,KONSTANTINIDIS K T,KLAPPENBACH J A,et al.DNA-DNA hybridization values and their relationship to whole-genome sequence similarities[J].International Journal of Systematic and Evolutionary Microbiology,2007,57(Pt 1):81-91.  RICHTER M,ROSSELLÓ-MÓRA R.Shifting the genomic gold standard for the prokaryotic species definition[J].PNAS,2009,106(45):19 126-19 131.  MOROVIC W,HIBBERD A A,ZABEL B,et al.Genotyping by PCR and high-throughput sequencing of commercial probiotic products reveals composition biases[J].Frontiers in Microbiology,2016,7:1747.  INGLIN R C,MEILE L,STEVENS M J A.Clustering of pan- and core-genome of Lactobacillus provides novel evolutionary insights for differentiation[J].BMC Genomics,2018,19(1):284.  DURANTI S,MILANI C,LUGLI G A,et al.Evaluation of genetic diversity among strains of the human gut commensal Bifidobacterium adolescentis[J].Scientific Reports,2016,6:23971.  MILANI C,DURANTI S,LUGLI G A,et al.Comparative genomics of Bifidobacterium animalis subsp.lactis reveals a strict monophyletic bifidobacterial taxon[J].Applied and Environmental Microbiology,2013,79(14):4 304-4 315.  范铁男, 邹积宏,卢行安,等.基质辅助激光解吸电离飞行时间质谱技术作为常见益生菌菌种鉴定辅助工具的研究[J].中国微生态学杂志,2010,22(11):970-973. FAN T N,ZOU J H,LU X A,et al.MALDI-TOF mass spectrometry as an assistant tool for identification of common probiotics[J].Chinese Journal of Microecology,2010,22(11):970-973.  CHAN M S,MAIDEN M C J,SPRATT B G.Database-driven Multi Locus Sequence Typing (MLST) of bacterial pathogens[J].Bioinformatics,2001,17(11):1 077-1 083.  MAIDEN M C J,VAN RENSBURG M J J,BRAY J E,et al.MLST revisited:The gene-by-gene approach to bacterial genomics[J].Nature Reviews Microbiology,2013,11(10):728-736.  PIGHTLING A W,PETTENGILL J B,LUO Y,et al.Interpreting whole-genome sequence analyses of foodborne bacteria for regulatory applications and outbreak investigations[J].Frontiers in Microbiology,2018,9:1482.