Bifidobacterium animalis subsp. lactis genome resources and metabolite profiling at the strain level and their ability to alleviate anxiety-like behavior in a sleep-deprived mouse model

Abstract

Bifidobacterium animalis subsp. lactis is a well-known probiotic with potential benefits for alleviating sub-health symptoms, including immune dysfunction and anxiety. Given the strain-specific nature of its probiotic effects, identifying effective strains for sub-health alleviation is crucial. In this study, we characterized 16 B animalis subsp. lactis isolates from fecal samples and probiotic sources. We assessed the genotype-phenotype correlations related to growth, carbon source utilization, and stress tolerance in vitro. Subsequently, we profiled 107 metabolites (including 28 alcohols and 17 esters) and quantified the levels of short-chain fatty acids and three other organic acids. Three B. animalis strains, GOLDGUT-BB21, WLBA7, and WLBA6, were selected and evaluated in a sleep-deprived mouse model. In vivo, WLBA3 reduced inflammation and oxidative stress by inhibiting the NLRP3 inflammasome pathway and modulating gut microbiota (e.g., Lactobacillus and Alistipes), which in turn significantly improved weight gain and fatigue resistance, attenuated cognitive function, and anxiety-like behavior. These findings provide insights into the diversity of B. animalis subsp. lactis strain resources and highlight the potential of WLBA3 as a candidate for alleviating sub-health symptoms.

Keywords

Bifidobacterium animalis subsp. Lactis / Probiotic strains / Metabolite profiling / Sleep-deprived mouse model / Anxiety-like behavior

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Given their roles as Executive Guest Editor and Guest Editor, respectively, Dr. Shuangjiang Liu and Dr. Chang Liu had no involvement in the peer review of this article, and had no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Dr Mudasir Dar.

Yan-Yi Zheng, Si-Lu Zhang, and Guo-Xun Xiao are staff members of the WONDERLAB Innovation Centre for Healthcare, an organization which is dedicated to the innovation and commercialization of probiotics and nutrition supplements and which has also partly supported this research. The funders had no role in the decision to publish this study.

CRediT authorship contribution statement

Yao-Kun Zhang: Writing - original draft, Visualization, Validation, Resources, Investigation, Formal analysis, Data curation. Liang Zhang: Writing - original draft, Visualization, Investigation, Funding acquisition, Formal analysis, Data curation. Xue Ni: Investigation, Data curation. Shu-Wen Zhang: Investigation, Data curation. Min-Zhi Jiang: Methodology, Investigation, Data curation. Si-Lu Zhang: Supervision, Conceptualization. Guo-Xun Xiao: Supervision, Conceptualization. He Jiang: Resources. Ming-Xia Bi: Supervision, Methodology. Yu-Lin Wang: Supervision, Formal analysis. Chang Liu: Supervision, Conceptualization. Shuang-Jiang Liu: Writing - review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

Data Availability Statement

The genomic data in this study were is available at the National Microbiology Data Center (https://nmdc.cn/resource/genomics), with Project Accession Number NMDC10019713.

Acknowledgments

This research is supported by National Key Research and Development Program of China (No. 2022YFA1304103). We thank Yan-Yi Zheng at the WONDERLAB Innovation Center for Healthcare for administrative assistance. We also thank Chang Xu and Li-Juan Feng at Shandong University for their support on experiments and providing bacterial strains.

References

[1] G. Li, F. Xie, S. Yan, X. Hu, B. Jin, J. Wang, J. Wu, D. Yin, Q. Xie Subhealth: definition, criteria for diagnosis and potential prevalence in the central region of China BMC Public Health, 13 (2013), p. 446

[2] R. Zhao, Y. Cai, X. Shao, B. Ma Improving the activity of Lycium barbarum polysaccharide on sub-health mice Food Funct., 6 (2015), pp. 2033-2040

[3] S. Feng, W. Liu, S. Zuo, T. Xie, H. Deng, Q. Zhang, B. Zhong Impaired function of the intestinal barrier in a novel sub-health rat model Mol. Med. Rep., 13 (2016), pp. 3459-3465

[4] M.L. Shi, M.Q. Yan, J. Su, J.J. Yu, S.Y. Ye, M. Fu, X.L. Hu, Z.W. Niu, W.Y. Wu, S.M. Chen, S.H. Chen, J.Z. Chen, G.Y. Lv Effects of dendrobium officinale ultrafine powder on sub-health mice induced by unhealthy lifestyle based on neuroendocrine immune system Food Funct., 13 (2022), pp. 12436-12450

[5] Y. Liu, P. Ge, X. Zhang, Y. Wu, Z. Sun, Q. Bai, S. Jing, H. Zuo, P. Wang, J. Cong, X. Li, K. Liu, Y. Wu, B. Wei Intrarelationships between suboptimal health status and anxiety symptoms: a network analysis J. Affect. Disord., 354 (2024), pp. 679-687

[6] G.H. Wang, Q. Wang, J.H. Xue, X.Q. Feng, X.Y. Xu, X.Y. Zhang, Y.L. jia, Z.W. Nie Current status and progress of sub-health interventions World J. Integr. Tradit. West. Med., 5 (2010), pp. 908-910

[7] M. Zhang, Z. Zhou, X. Tao, L. Huang, E. Zhu, L. Yu, H. Liu Prevalence of subhealth status and its effects on mental health and smartphone addiction: a cross-sectional study among Chinese medical students Rev. Assoc. Med. Bras., 68 (2022) (1992), pp. 222-226

[8] G. Mahara, X.Tan, W. Wang Ris k factors, health status, and Risk groups in suboptimal health condition W. Wang (Ed.),All Around Suboptimal Health: Advanced Approaches By Predictive, Preventive and Personalised Medicine For Healthy Populations , Springer Nature Switzerland, Cham (2024), pp. 61-72

[9] C. Ma, W. Xu, L. Zhou, S. Ma, Y. Wang Association between lifestyle factors and suboptimal health status among Chinese college freshmen: a cross-sectional study BMC Public Health, 18 (2018), p. 105

[10] J. Zhao, X. Shen, H.X. Liu, Y.R. Guo, G.Q. Gao, Z.H. Sun Research progress of probiotics in alleviating sub-health related symptoms J. Chin. Inst. Food Sci. Technol., 24 (2024), pp. 432-440

[11] M. Jungersen, A. Wind, E. Johansen, J.E. Christensen, B. Stuer-Lauridsen, D. Eskesen The science behind the probiotic strain bifidobacterium animalis subsp. Lactis BB-12((R)) Microorganisms, 2 (2014), pp. 92-110

[12] J. Cheng, A. Laitila, A.C. Ouwehand Bifidobacterium animalis subsp. Lactis HN019 effects on Gut health: a review Front. Nutr., 8 (2021), Article 790561

[13] H.M. Uusitupa, P. Rasinkangas, M.J. Lehtinen, S.M. Makela, K. Airaksinen, H. Anglenius, A.C. Ouwehand, J. Maukonen Bifidobacterium animalis subsp. Lactis 420 for Metabolic Health: review of the research Nutrients , 12 (2020), p. 892

[14] B. Sun, T. Ma, Y. Li, N. Yang, B. Li, X. Zhou, S. Guo, S. Zhang, L.Y. Kwok, Z. Sun, H. Zhang Bifidobacterium lactis probio-M8 adjuvant treatment confers added benefits to patients with coronary artery disease via target modulation of the gut-heart/-brain axes mSystems, 7 (2022), Article e0010022

[15] N.P. West, L. Hughes, R. Ramsey, P. Zhang, C.J. Martoni, G.J. Leyer, A.W. Cripps, A.J. Cox Probiotics, anticipation stress, and the acute immune response to night shift Front. Immunol., 11 (2020), Article 599547

[16] M.Le Barz, N. Daniel, T.V. Varin, S. Naimi, V. Demers-Mathieu, G. Pilon, J. Audy, E. Laurin, D. Roy, M.C. Urdaci, D. St-Gelais, I. Fliss, A. Marette In vivo screening of multiple bacterial strains identifies Lactobacillus rhamnosus Lb102 and bifidobacterium animalis ssp. Lactis Bf141 as probiotics that improve metabolic disorders in a mouse model of obesity FASEB J., 33 (2019), pp. 4921-4935

[17] T. Seemann Prokka: rapid prokaryotic genome annotation Bioinformatics, 30 (2014), pp. 2068-2069

[18] A.J. Page, C.A. Cummins, M. Hunt, V.K. Wong, S. Reuter, M.T. Holden, M. Fookes, D. Falush, J.A. Keane, J. Parkhill Roary: rapid large-scale prokaryote pan genome analysis Bioinformatics, 31 (2015), pp. 3691-3693

[19] I. Letunic, P. Bork Interactive Tree of Life (iTOL): an online tool for phylogenetic tree display and annotation Bioinformatics, 23 (2007), pp. 127-128

[20] J.F. Wardman, S.G. Withers Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening RSC Chem. Biol., 5 (2024), pp. 595-616

[21] A.G. McArthur, N. Waglechner, F. Nizam, A. Yan, M.A. Azad, A.J. Baylay, K. Bhullar, M.J. Canova, G. De Pascale, L. Ejim, L. Kalan, A.M. King, K. Koteva, M. Morar, M.R. Mulvey, J.S. O'Brien, A.C. Pawlowski, L.J. Piddock, P. Spanogiannopoulos, A.D. Sutherland, I. Tang, P.L. Taylor, M. Thaker, W. Wang, M. Yan, T. Yu, G.D. Wright The comprehensive antibiotic resistance database Antimicrob. Agents Chemother., 57 (2013), pp. 3348-3357

[22] W.H. Traub, B. Leonhard, D. Bauer Antibiotic susceptibility of Stenotrophomonas (Xanthomonas) maltophilia: comparative (NCCLS criteria) evaluation of antimicrobial drugs with the agar dilution and the agar disk diffusion (Bauer-Kirby) tests Chemother. (Angel.), 44 (1998), pp. 164-173

[23] S.E. Gilliland, T.E.Staley, L.J. Bush Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct J. Dairy. Sci., 67 (1984), pp. 3045-3051

[24] R. Abdugheni, W.Z. Wang, Y.J. Wang, M.X. Du, F.L. Liu, N. Zhou, C.Y. Jiang, C.Y. Wang, L. Wu, J. Ma, C. Liu, S.J. Liu Metabolite profiling of human-originated lachnospiraceae at the strain level Imeta, 1 (2022), p. e58

[25] L.W. Sumner, A. Amberg, D. Barrett, M.H. Beale, R. Beger, C.A. Daykin, T.W. Fan, O. Fiehn, R. Goodacre, J.L. Griffin, T. Hankemeier, N. Hardy, J. Harnly, R. Higashi, J. Kopka, A.N. Lane, J.C. Lindon, P. Marriott, A.W. Nicholls, M.D. Reily, J.J. Thaden, M.R. Viant Proposed minimum reporting standards for chemical analysis Chemical analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI) Metabolomics, 3 (2007), pp. 211-221

[26] L. Chen, Q. Yue, X. Zhang, M. Xiang, C. Wang, S. Li, Y. Che, F.J. Ortiz-Lopez, G.F. Bills, X. Liu, Z. An Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis BMC Genom., 14 (2013), p. 339

[27] D. Liu, L.S. Xie, S. Lian, K. Li, Y. Yang, W.Z. Wang, S. Hu, S.J. Liu, C. Liu, Z. He Anaerostipes hadrus, a butyrate-producing bacterium capable of metabolizing 5-fluorouracil mSphere, 9 (2024), Article e0081623

[28] M. Hall, R.G. Beiko 16S rRNA gene analysis with QIIME2 Methods Mol. Biol., 1849 (2018), pp. 113-129

[29] F. Lyu, F. Han, C. Ge, W. Mao, L. Chen, H. Hu, G. Chen, Q. Lang, C. Fang OmicStudio: a composable bioinformatics cloud platform with real-time feedback that can generate high-quality graphs for publication Imeta, 2 (2023), p. e85

[30] C. Liu, M.X. Du, R. Abuduaini, H.Y. Yu, D.H. Li, Y.J. Wang, N. Zhou, M.Z. Jiang, P.X. Niu, S.S. Han, H.H. Chen, W.Y. Shi, L. Wu, Y.H. Xin, J. Ma, Y. Zhou, C.Y. Jiang, H.W. Liu, S.J. Liu Enlightening the taxonomy darkness of human gut microbiotas with a cultured biobank Microbiota, 9 (2021), p. 119

[31] A.K. Martins, F.S. Martins, D.A. Gomes, S.D. Elian, A.T. Vieira, M.M. Teixeira, D.C. Cara, R.M. Nardi, J.R. Nicoli Evaluation of in vitro antagonism and of in vivo immune modulation and protection against pathogenic experimental challenge of two probiotic strains of bifidobacterium animalis var. Lactis Arch. Microbiol., 192 (2010), pp. 995-1003

[32] D. Eskesen, L. Jespersen, B. Michelsen, P.J. Whorwell, S. Muller-Lissner, C.M. Morberg Effect of the probiotic strain bifidobacterium animalis subsp. Lactis, BB-12(R), on defecation frequency in healthy subjects with low defecation frequency and abdominal discomfort: a randomised, double-blind, placebo-controlled, parallel-group trial Br. J. Nutr, 114 (2015), pp. 1638-1646

[33] A. Ibarra, M. Latreille-Barbier, Y. Donazzolo, X. Pelletier, A.C. Ouwehand Effects of 28-day bifidobacterium animalis subsp. Lactis HN019 supplementation on colonic transit time and gastrointestinal symptoms in adults with functional constipation: a double-blind, randomized, placebo-controlled, and dose-ranging trial Gut. Microbes., 9 (2018), pp. 236-251

[34] E. Dimidi, A. Zdanaviciene, S. Christodoulides, S. Taheri, P. Louis, P.I. Duncan, N. Emami, R. Crabbe, C.A. De Castro, P. McLean, G.E. Bergonzelli, K. Whelan, S. Mark Scott Randomised clinical trial: bifidobacterium lactis NCC2818 probiotic vs placebo, and impact on gut transit time, symptoms, and gut microbiology in chronic constipation Aliment. Pharmacol. Ther., 49 (2019), pp. 251-264

[35] M. Del Piano, S. Carmagnola, A. Anderloni, S. Andorno, M. Ballare, M. Balzarini, F. Montino, M. Orsello, M. Pagliarulo, M. Sartori, R. Tari, F. Sforza, L. Capurso The use of probiotics in healthy volunteers with evacuation disorders and hard stools: a double-blind, randomized, placebo-controlled study J. Clin. Gastroenterol., 44 (Suppl 1) (2010), pp. S30-S34

[36] S. Han, Y. Lu, J. Xie, Y. Fei, G. Zheng, Z. Wang, J. Liu, L. Lv, Z. Ling, B. Berglund, M. Yao, L. Li Probiotic gastrointestinal transit and colonization after oral administration: a long journey Front. Cell Infect. Microbiol., 11 (2021), Article 609722

[37] M. Govaert, C. Rotsaert, C. Vannieuwenhuyse, C. Duysburgh, S. Medlin, M. Marzorati, H. Jarrett Survival of probiotic bacterial cells in the upper gastrointestinal tract and the effect of the surviving population on the colonic microbial community activity and composition Nutrients, 16 (2024), p. 2791

[38] K. Krastel, D.B. Senadheera, R. Mair, J.S. Downey, S.D. Goodman, D.G. Cvitkovitch Characterization of a glutamate transporter operon, glnQHMP, in Streptococcus mutans and its role in acid tolerance J. Bacteriol., 192 (2010), pp. 984-993

[39] Y. Shinde, I. Ahmad, S. Surana, H. Patel The mur enzymes chink in the armour of mycobacterium tuberculosis cell wall Eur. J. Med. Chem., 222 (2021), Article 113568

[40] K.M. Cho, Y.S. Kim, M. Lee, H.Y. Lee, Y.S. Bae Isovaleric acid ameliorates ovariectomy-induced osteoporosis by inhibiting osteoclast differentiation J. Cell Mol. Med., 25 (2021), pp. 4287-4297

[41] I. Mukhopadhya, P. Louis, Gut microbiota-derived short-chain fatty acids and their role in human health and disease, (2025).

[42] Z. Ju, J. Shao, M. Zhou, J. Jin, H. Pan, P. Ding, R. Huang Transcriptomic and metabolomic profiling reveal the p53-dependent benzeneacetic acid attenuation of silica-induced epithelial-mesenchymal transition in human bronchial epithelial cells Cell Biosci., 11 (2021), p. 30

[43] M. Zielinska-Blajet, J. Feder-Kubis Monoterpenes and their derivatives-recent development in biological and medical applications Int. J. Mol. Sci., 21 (2020), p. 7078

[44] X. Yin, W. Liu, H. Feng, J. Huang, Q. Wang, Q. Zhang, J. He, R. Wang Bifidobacterium animalis subsp. Lactis A6 attenuates hippocampal damage and memory impairments in an ADHD rat model Food Funct., 15 (2024), pp. 2668-2678

[45] R. Ramachandran, A. Manan, J. Kim, S. Choi NLRP3 inflammasome: a key player in the pathogenesis of life-style disorders Exp. Mol. Med., 56 (2024), pp. 1488-1500

[46] L. Zhang, S. Zhang, M. Jiang, X. Ni, M. Du, H. Jiang, M. Bi, Y. Wang, C. Liu, S. Liu Limosilactobacillus reuteri alleviates anxiety-like behavior and intestinal symptoms in two stressed mouse models Nutrients, 16 (2024), p. 3209

[47] W. Zhang, W. Qu, H. Wang, H. Yan Antidepressants fluoxetine and amitriptyline induce alterations in intestinal microbiota and gut microbiota function in rats exposed to chronic unpredictable mild stress Transl. Psychiatry, 11 (2021), p. 131

[48] M.F. Khan, C.D. Murphy Bacterial degradation of the anti-depressant drug fluoxetine produces trifluoroacetic acid and fluoride ion Appl. Microbiol. Biotechnol., 105 (2021), pp. 9359-9369