The prevailing national standard for probiotic enumeration falls short in achieving strain-level identification and quantification within complex biological matrices, posing a notable technical hurdle that constrains current industry regulations and impedes product differentiation.To address this issue, this study utilized Lactobacillus rhamnosus MP108 as a model and devised a strain-specific quantitative approach leveraging whole-genome data of the strain, targeting characteristic genetic sequences, and employing advanced techniques such as real-time fluorescence quantitative PCR.The findings demonstrated that this method effectively discerned the presence of the MP108 strain and enabled precise quantification in complex microbial environments, boasting a detection threshold of 104 cells per gram of biological sample and a sensitivity of 10 copies per microliter of DNA solution.Notably, the quantitative accuracy of this approach in complex systems closely aligned with reference values obtained through conventional plate counting methods utilizing pure culture strains.Consequently, this method holds promise in furnishing foundational insights for enhancing the nuanced detection of probiotic foods.
WANG Jingyu
,
QIAN Xin
,
JIN Zhiying
,
TIAN Peijun
,
ZHAI Qixiao
. Establishment of a strain-level quantification method for Lactobacillus rhamnosus MP108 in complex biological systems[J]. Food and Fermentation Industries, 2025
, 51(6)
: 288
-293;299
.
DOI: 10.13995/j.cnki.11-1802/ts.040099
[1] MUKHERJI A, KOBIITA A, YE T, et al.Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs [J].Cell, 2013, 153(4):812-827.
[2] HONDA K, LITTMAN D R.The microbiota in adaptive immune homeostasis and disease[J].Nature, 2016, 535(7610):75-84.
[3] AHRENS A P, HYÖTYLÄINEN T, PETRONE J R, et al.Infant microbes and metabolites point to childhood neurodevelopmental disorders[J].Cell, 2024, 187(8):1853-1873:e15.
[4] TAN S, SANTOLAYA J L, WRIGHT T F, et al.Interaction between the gut microbiota and colonic enteroendocrine cells regulates host metabolism[J].Nature Metabolism, 2024, 6(6):1076-1091.
[5] JERYE K, LÜKEN H, STEFFEN A, et al.Activity-based protein profiling identifies protein disulfide-isomerases as target proteins of the volatile salinilactones[J].Advanced Science, 2024, 11(18):e2309515.
[6] JOHNSON J S, SPAKOWICZ D J, HONG B Y, et al.Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis[J].Nature Communications, 2019, 10(1):5029.
[7] 胡陆军. 复杂样品中双歧杆菌的高通量检测和菌种高效分离方法的研究[D]. 无锡: 江南大学, 2017.
HU L J. Study on Qualcomm detection of Bifidobacterium in complex samples and efficient separation of strains[D]. Wuxi: Jiangnan University, 2017.
[8] JAIN C, RODRIGUEZ-R L M, PHILLIPPY A M, et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J].Nature Communications, 2018, 9(1):5114.
[9] KIRSCH J M, HRYCKOWIAN A J, DUERKOP B A.A metagenomics pipeline reveals insertion sequence-driven evolution of the microbiota[J].Cell Host & Microbe, 2024, 32(5):739-754.e4.
[10] STÄMMLER F, GLÄSNER J, HIERGEIST A, et al.Adjusting microbiome profiles for differences in microbial load by spike-in bacteria[J].Microbiome, 2016, 4(1):28.
[11] SEEMANN T.Prokka:Rapid prokaryotic genome annotation[J].Bioinformatics, 2014, 30(14):2068-2069.
[12] ZHAO Y B, WU J Y, YANG J H, et al.PGAP:Pan-genomes analysis pipeline[J].Bioinformatics, 2012, 28(3):416-418.
[13] PRICE M N, DEHAL P S, ARKIN A P.FastTree 2:Approximately maximum-likelihood trees for large alignments[J].PLoS One, 2010, 5(3):e9490.
[14] LI L, STOECKERT C JJr, ROOS D S.OrthoMCL:Identification of ortholog groups for eukaryotic genomes[J].Genome Research, 2003, 13(9):2178-2189.
[15] GRANT J R, ENNS E, MARINIER E, et al.Proksee:In-depth characterization and visualization of bacterial genomes[J].Nucleic Acids Research, 2023, 51(W1):W484-W492.
[16] ZHENG J S, WITTOUCK S, SALVETTI E, et al.A taxonomic note on the genus Lactobacillus:Description of 23 novel Genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae[J].International Journal of Systematic and Evolutionary Microbiology, 2020, 70(4):2782-2858.
[17] HORESH G, TAYLOR-BROWN A, MCGIMPSEY S, et al.Different evolutionary trends form the twilight zone of the bacterial pan-genome[J].Microbial Genomics, 2021, 7(9):000670.
[18] BARRATT M J, NUZHAT S, AHSANK, et al. Bifidobacterium infantis treatment promotes weight gain in Bangladeshi infants with severe acute malnutrition[J].Science Translational Medicine, 2022, 14(640):eabk1107.
[19] XIAO Y, WANG C, ZHAO J X, et al.Quantitative detection of Bifidobacterium longum strains in feces using strain-specific primers[J].Microorganisms, 2021, 9(6):1159.
