Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarum CRM56-2 Isolated from Fermented Tea Leaves

Main Article Content

Engkarat Kingkaew
Naoto Tanaka
Yuh Shiwa
Jaruwan Sitdhipol
Rattanatda Nuhwa
Somboon Tanasupawat

Abstract

Lactiplantibacillus plantarum is a widely studied species known for its probiotic properties that can help alleviate serum cholesterol levels. Whole-genome sequencing provides genetic information on probiotic attributes, metabolic activities and safety assessment. This study investigates the probiotic properties of strain CRM56-2, isolated from Thai fermented tea leaves, using Whole-Genome Sequencing (WGS) to evaluate the safety, health-promoting genes and functional analysis. Strain CRM56-2 showed bile salt hydrolase (BSH) activity, assimilated cholesterol at a rate of 75.94%, tolerated acidic and bile environments and attached to Caco-2 cells. Based on ANIb (98.9%), ANIm (99.2%), and digital DNA–DNA hybridisation (98.3%), strain CRM56-2 was identified as L. plantarum. In silico analysis revealed that it was not pathogenic and contained no antibiotic-resistance genes or plasmids. L. plantarum CRM56-2 possessed genes linked to several probiotic properties and beneficial impacts. The genome of strain CRM56-2 suggested that L. plantarum CRM56-2 is non-hazardous, with potential probiotic characteristics and beneficial impacts, which could enhance its probiotic application. Consequently, L. plantarum CRM56-2 demonstrated excellent cholesterol-lowering activity and probiotic properties. 

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Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarum CRM56-2 Isolated from Fermented Tea Leaves. (2024). Tropical Life Sciences Research, 35(2), 249–269. https://doi.org/10.21315/tlsr2024.35.2.12
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References

Abriouel H, Pérez Montoro B, Casimiro-Soriguer C S, Pérez Pulido A J, Knapp C W, Caballero Gómez N, Castillo-Gutiérrez S, Estudillo-Martínez M D, Gálvez A and Benomar N. (2017). Insight into potential probiotic markers predicted in Lactobacillus pentosus MP-10 genome sequence. Frontiers in Microbiology 8: 891. https://doi.org/10.3389/fmicb.2017.00891

Alayande K A, Aiyegoro O A, Nengwekhulu T M, Katata-Seru L and Ateba C N. (2020). Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1. PLoS One 15: e0235873. https://doi.org/10.1371/journal.pone.0235873

Arasu M V, Al-Dhabi N A, Ilavenil S, Choi K C and Srigopalram S. (2016). In vitro importance of probiotic Lactobacillus plantarum related to medical field. Saudi Journal of Biological Sciences 23(1): 6–10. https://doi.org/10.1016/j.sjbs.2015.09.022

Arndt D, Grant J R, Marcu A, Sajed T, Pon A, Liang Y and Wishart D S. (2016). PHASTER: A better, faster version of the PHAST phage search tool. Nucleic Acids Research 44: 16–21. https://doi.org/10.1093/nar/gkw387

Aziz R K, Bartels D, Best A A, DeJongh M, Disz, T, Edwards R A, Formsma K, Gerdes S, Glass E M, Kubal M and Meyer F. (2008). The RAST Server: Rapid annotations using subsystems technology. BMC Genomics 9(1): 1–15. https://doi.org/10.1186/1471-2164-9-75

Bortolaia V, Kaas R S, Ruppe E, Roberts M C, Schwarz S, Cattoir V, Philippon A, Allesoe R L, Rebelo A R, Florensa A F and Fagelhauer L. (2020). ResFinder 4.0 for predictions of phenotypes from genotypes. Journal of Antimicrobial Chemotherapy 75(12): 3491–3500. https://doi.org/10.1093/jac/dkaa345

Botta C, Alberto A, Anna G, Lorenzo B, Marta B, Luca C, and Kalliopi R. (2017). Genomic assessment in Lactobacillus plantarum links the butyrogenic pathway with glutamine metabolism. Scientific Reports 7(1): 15975. https://doi.org/10.1038/s41598-017-16186-8

Bustos I, Garcia-Cayuela T, Hernandez-Ledesma B, Pelaez C, Requena T and Martínez-Cuesta M C. (2012). Effect of flavan-3-ols on the adhesion of potential probiotic lactobacilli to intestinal cells. Journal of Agricultural and Food Chemistry 60(36): 9082–9088. https://doi.org/10.1021/jf301133g

Cantarel B L, Coutinho P M, Rancurel C, Bernard T, Lombard V and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Research 37: 233–238. https://doi.org/10.1093/nar/gkn663

Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, Møller Aarestrup F and Hasman H. (2014). In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrobial Agents and Chemotherapy 58(7): 3895–3903. https://doi.org/10.1128/AAC.02412-14

Chun J, Oren A, Ventosa A, Christensen H, Arahal D R, da Costa M S, Rooney A P, Yi H, Xu X W, De Meyer S and Trujillo M E. (2018). Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. International Journal of Systematic and Evolutionary Microbiology 68(1): 461–466. https://doi.org/10.1099/ijsem.0.002516

Cosentino S, Voldby Larsen M, Møller Aarestrup F and Lund O. (2013). PathogenFinderdistinguishing friend from foe using bacterial whole genome sequence data. PloS One 8(10): e77302. https://doi.org/10.1371/journal.pone.0077302

Cowan S T and Steel K J. (1965). Manual for the identification of medical bacteria. New York: Cambridge University Press.

Davis J J, Wattam A R, Aziz R K, Brettin T, Butler R, Butler R M, Chlenski P, Conrad N, Dickerman A, Dietrich E M and Gabbard J L. (2020). The PATRIC Bioinformatics Resource Center: Expanding data and analysis capabilities. Nucleic Acids Research 48(D1): 606–612. https://doi.org/10.1093/nar/gkz943

De Jesus L C L, Aburjaile F F, Sousa T D J, Felice A G, Soares S D C, Alcantara L C J and Azevedo V A D C. (2022). Genomic characterization of Lactobacillus delbrueckii strains with probiotics properties. Frontiers in Bioinformatics 2: 912795. https://doi.org/10.3389/fbinf.2022.912795

Diale M O, Kayitesi E and Serepa-Dlamini M H. (2021). Genome in silico and in vitro analysis of the probiotic properties of a bacterial endophyte, Bacillus paranthracis strain mhsd3. Frontiers in Genetics 12: 672149. https://doi.org/10.3389/fgene.2021.672149

FAO/WHO.(2002). Guidelines for the evaluation of probiotics in food. FAO/WHO Working Group 1–11.

Guinane C M, Crispie F and Cotter P D. (2016). Value of microbial genome sequencing for probiotic strain identification and characterization: Promises and pitfalls. In N Hyland and C Stanton (eds.), The gut-brain axis. Academic Press, 45–60. https://doi.org/10.1016/B978-0-12-802304-4.00004-9

Hyronimus B, Le Marrec C, Sassi A H and Deschamps A. (2000). Acid and bile tolerance of spore-forming lactic acid bacteria. International Journal of Food Microbiology 61(2–3): 193–197. https://doi.org/10.1016/S0168-1605(00)00366-4

Kanehisa M, Sato Y and Morishima K. (2016). BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. Journal of Molecular Biology 428(4): 726–731. https://doi.org/10.1016/j.jmb.2015.11.006

Kingkaew E, Konno H, Hosaka, Y and Tanasupawat S. (2022). Probiogenomic analysis of Lactiplantibacillus sp. LM14-2 from fermented mussel (Hoi-Dong), and evaluation of its cholesterol-lowering and immunomodulation effects. Probiotics and Antimicrobial Proteins 1–15. https://doi.org/10.1007/s12602-022-09977-7

Kingkaew E, Woraprayote W, Booncharoen A, Niwasabutra, K., Janyaphisan T, Vilaichone, Ratha-Korn V, Yoshio Y, Wonnop V and Tanasupawat S. (2023). Functional genome analysis and anti-Helicobacter pylori activity of a novel bacteriocinogenic Lactococcus sp. NH2-7C from Thai fermented pork (Nham). Scientific Reports 13(1): 20362. https://doi.org/10.1038/s41598-023-47687-4

Lane D J. (1991). 16S/23S rRNA sequencing. In E Stackebrandt and M Goodfellow (eds.), Nucleic acid techniques in bacterial systematic. New York: John Wiley and Sons, 115–175.

Le B and Yang S H. (2018). Efficacy of Lactobacillus plantarum in prevention of inflammatory bowel disease. Toxicology Reports 5: 314–317. https://doi.org/10.1016/j.toxrep.2018.02.007

Li B, Jin D, Etareri Evivie S, Li N, Yan F, Zhao L, Liu F and Huo G. (2017). Safety assessment of Lactobacillus helveticus KLDS1. 8701 based on whole genome sequencing and oral toxicity studies. Toxins 9(10): 301. https://doi.org/10.3390/toxins9100301

Martino M E, Bayjanov J R, Caffrey B E, Wels M, Joncour P, Hughes S., Gillet, B., Kleerebezem M, van Hijum S A and Leulier F. (2016). Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats. Environmental Microbiology 18(12): 4974–4989. https://doi.org/10.1111/1462-2920.13455

Mazmanian S K, Round J L and Kasper D L. (2008). A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453(7195): 620–625. https://doi.org/10.1038/nature07008

Meier-Kolthoff J P, Auch A F, Klenk H P and Göker M. (2013). Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 1–14. https://doi.org/10.1186/1471-2105-14-60

Meier-Kolthoff J P and Göker M. (2019). TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nature Communications 10(1): 2182. https://doi.org/10.1038/s41467-019-10210-3

Okada S, Toyoda T and Kozaki M. (1978). An easy method for the determination of the optical types of lactic acid produced by lactic acid bacteria. Agricultural and Biological Chemistry 42(9): 1781–1783. https://doi.org/10.1080/00021369.1978.10863246

Parks D H, Imelfort M, Skennerton C T, Hugenholtz P and Tyson G W. (2015). CheckM: Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Research 25(7): 1043–1055. https://doi.org/10.1101/gr.186072.114

Pei Z, Sadiq F A, Han X, Zhao J, Zhang H, Ross R P, Lu W and Chen W. (2021). Comprehensive scanning of prophages in Lactobacillus: Distribution, diversity, antibiotic resistance genes, and linkages with CRISPR-Cas systems. Msystems 6(3): e01211–20. https://doi.org/10.1128/mSystems.01211-20

Pereira D I and Gibson G R. (2002). Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Applied and Environmental Microbiology 68(9): 4689–4693. https://doi.org/10.1128/AEM.68.9.4689-4693.2002

Phuengjayaem S, Phinkian N, Tanasupawat S and Teeradakorn S. (2017). Diversity and succinic acid production of lactic acid bacteria isolated from animals, soils and tree barks. Research Journal of Microbiology 12: 177–186. https://doi.org/10.3923/jm.2017.177.186

Richter M and Rosselló-Móra R. (2009). Shifting the genomic gold standard for the prokaryotic species definition. Proceedings of the National Academy of Sciences 106(45): 19126–19131. https://doi.org/10.1073/pnas.0906412106

Richter M, Rosselló-Móra R, Oliver Glöckner F and Peplies J. (2016). JSpeciesWS: A web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32(6): 929–931. https://doi.org/10.1093/bioinformatics/btv681

Rudel L L and Morris M D. (1973). Determination of cholesterol using o-phthalaldehyde. Journal of Lipid Research 14(3): 364–366. https://doi.org/10.1016/S0022-2275(20)36896-6

Siezen R J, Tzeneva V A, Castioni A, Wels M, Phan H T, Rademaker J L, Starrenburg, M J, Kleerebezem M, Molenaar D and van Hylckama Vlieg J E. (2010). Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. Environmental Microbiology 12(3): 758–773. https://doi.org/10.1111/j.1462-2920.2009.02119.x

Stothard P, Grant J R and Van Domselaar G. (2019). Visualizing and comparing circular genomes using the CGView family of tools. Briefings in Bioinformatics 20(4): 1576–1582. https://doi.org/10.1093/bib/bbx081

Surachat K, Sangket U, Deachamag P and Chotigeat W. (2017). In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases. PLoS One 12(8): e0183548. https://doi.org/10.1371/journal.pone.0183548

Surve S, Shinde D B and Kulkarni R. (2022). Isolation, characterization and comparative genomics of potentially probiotic Lactiplantibacillus plantarum strains from Indian foods. Scientific Reports 12(1): 1940. https://doi.org/10.1038/s41598-022-05850-3

Tanasupawat S, Thongsanit J, Okada S and Komagata K. (2002). Lactic acid bacteria isolated from soy sauce mash in Thailand. The Journal of General and Applied Microbiology 48(4): 201–209. https://doi.org/10.2323/jgam.48.201

Tanizawa Y, Fujisawa T and Nakamura Y. (2018). DFAST: A flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 34(6): 1037–1039. https://doi.org/10.1093/bioinformatics/btx713

Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki E P, Zaslavsky L, Lomsadze A, Pruitt K D, Borodovsky M and Ostell J. (2016). NCBI prokaryotic genome annotation pipeline. Nucleic Acids Research 44(14): 6614–6624. https://doi.org/10.1093/nar/gkw569

Tomaro-Duchesneau C, Jones M L, Shah D, Jain P, Saha S and Prakash S. (2014). Cholesterol assimilation by Lactobacillus probiotic bacteria: An in vitro investigation. BioMed Research International 2014. https://doi.org/10.1155/2014/380316

van den Nieuwboer M, van Hemert S, Claassen E and de Vos W M. (2016). Lactobacillus plantarum WCFS 1 and its host interaction: A dozen years after the genome. Microbial Biotechnology 9(4): 452–465. https://doi.org/10.1111/1751-7915.12368

Yamada K and Komagata K. (1970). Taxonomic studies on coryneform bacteria III. DNA base composition of coryneform bacteria. The Journal of General and Applied Microbiology 16(3): 215–224. https://doi.org/10.2323/jgam.16.3_215

Ye K, Li P and Gu Q. (2020). Complete genome sequence analysis of a strain Lactobacillus pentosus ZFM94 and its probiotic characteristics. Genomics 112(5): 3142–3149. https://doi.org/10.1016/j.ygeno.2020.05.015

Yoon S H, Ha S M, Kwon S, Lim J, Kim Y, Seo H and Chun J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology 67(5): 1613. https://doi.org/10.1099/ijsem.0.001755

Zhang H, Yohe T, Huang L, Entwistle S, Wu P, Yang Z, Busk, P K, Xu Y and Yin Y. (2018). dbCAN2: A meta server for automated carbohydrate-active enzyme annotation. Nucleic Acids Research 46: 95–101. https://doi.org/10.1093/nar/gky418

Zhang N, Li C, Niu Z, Kang H, Wang M, Zhang B and Tian H. (2020). Colonization and immunoregulation of Lactobacillus plantarum BF_15, a novel probiotic strain from the feces of breast-fed infants. Food and Function 11(4): 3156–3166. https://doi.org/10.1039/C9FO02745A