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Trop Life Sci Res
Trop Life Sci Res
Tropical Life Sciences Research
Tropical Life Sciences Research
1985-3718
2180-4249
Penerbit Universiti Sains Malaysia

10.21315/tlsr2024.35.2.12
tlsr-35-2-249
Articles
Genomic Assessment of Potential Probiotic Lactiplantibacillus plantarum CRM56-2 Isolated from Fermented Tea Leaves
Kingkaew Engkarat Investigation Methodology Data curation Writing - review & editing 12
Tanaka Naoto Methodology Writing - review & editing 3
Shiwa Yuh Investigation Methodology Data curation 3
Sitdhipol Jaruwan Methodology Funding acquisition 4
Nuhwa Rattanatda Investigation Methodology Data curation Writing - original draft 1
Tanasupawat Somboon Supervision Funding acquisition Writing - review & editing 1*
1 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
3 Department of Molecular Microbiology, Faculty of Life Sciences, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
4 Biodiversity Research Centre, Research and Development Group for Bio-Industries, Thailand Institute of Scientific and Technological Research, Pathum Thani 12120, Thailand
* Corresponding author: somboon.t@chula.ac.th
7 2024
31 7 2024
35 2 249269
04 5 2023
19 2 2024
© Penerbit Universiti Sains Malaysia, 2024
2024
https://creativecommons.org/licenses/by/4.0/ This work is licensed under the terms of the Creative Commons Attribution (CC BY) (http://creativecommons.org/licenses/by/4.0/).
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.

Lactiplantibacillus plantarum
Fermented Tea Leaves
Genome Sequencing
Safety Evaluation
Probiotic Properties
Cholesterol-Lowering Activity
Thailand Science research and Innovation Fund Chulalongkorn UniversityHEA663300001
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pmcHighlights

Strain CRM56-2 isolated from fermented tea leaves was identified as Lactiplantibacillus plantarum based on ANIb (98.9%), ANIm (99.2%), and digital DNA–DNA hybridisation (98.3%).

L. plantarum 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 genomic analysis, L. plantarum CRM56-2 possessed genes linked to several probiotic properties and health-promoting effects, which could enhance its probiotic application.

INTRODUCTION

Lactic acid bacteria (LAB) are commonly used as probiotics because of their desirable features, such as safety, and longevity in the gastrointestinal tract (GIT) (Ye et al. 2020). Lactobacillus plantarum is mostly versatile of the extensively studied species, and it is found in fermented meat and plant sources (Siezen et al. 2010). The ability of L. plantarum to survive gastric and bile and its ability to survive and propagate in the GIT (Le & Yang 2018; Zhang et al. 2020) make it a promising target for probiotic research. In addition, L. plantarum is receiving attention in pharmaceutical sciences due to its cholesterol-lowering properties (Arasu et al. 2016). Probiotics can lower serum cholesterol using prebiotics to synthesise short-chain fatty acids (SCFAs), which can inhibit hepatic cholesterol synthesis, decrease serum lipids, and directly assimilate cholesterol (Pereira & Gibson 2002).

Several L. plantarum strains show potential probiotic traits, including adaptable growing characteristics, stress endurance, potent GIT survival and physiological roles, such as cholesterol-lowering (van den Nieuwboer et al. 2016). However, safety assessment is crucial in food and health applications, considering the growing concerns regarding antibiotic resistance and virulence factors. Therefore, whole-genome sequencing (WGS) analysis enables precise identification while providing molecular information on probiotic attributes, putative metabolisms and safety evaluation, such as virulent genetic elements, antibiotic resistance genes (ARGs), as well as genetic factors associated with risky substances (Guinane et al. 2016; Li et al. 2017). With the publication of whole-genome sequences of several L. plantarum strains in the NCBI database, comprehensive knowledge of L. plantarum’s functional properties and innovative applications becomes achievable. The genomic analysis must define a probiotic characteristic of an interesting strain. Selection criteria were bile salt hydrolase (BSH) activity, the highest cholesterol assimilation ability, and one of the significant probiotic species. Furthermore, genomic characterisation of L. plantarum strains isolated from the Thai-fermented leaves of Camellia sinensis needs investigation.

Accordingly, the present study endeavored to assess both the cholesterol-lowering effects and probiotic properties of strain CRM56-2. Additionally, WGS was employed to appraise the safety and probiotic-associated genes of this strain CRM56-2.

MATERIALS AND METHODS

Isolation

Strain CRM56-2 was isolated from Camellia sinensis obtained from the Chiang Rai province in Thailand. The sample (0.5 g) was added in De Man, Rogosa and Sharpe (MRS) broth and incubated at 37°C for 48 h–72 h. A loopful of the culture was streaked on MRS agar plates containing 0.3% (w/v) CaCO3. A single colony surrounded by a clear zone was selected and purified on MRS agar plates. The pure culture was preserved in 10% skim milk at −80°C and lyophilised.

Identification

Phenotypic characteristics

The strain’s cell shape, size, arrangement and colonial appearance were observed by growing cells on MRS agar plates for two days. Gram staining was performed following the procedure described by Cowan and Steel (1965). The activity of catalase, reduction of nitrate, gas formation, hydrolysis of arginine, aesculin production, as well as slime formation were determined using the methods previously reported by Tanasupawat et al. (2002). Growth under different pH (3.5–10.0), temperatures (10, 15, 30, 37, 40, 42 and 45°C), and NaCl concentrations (1, 3, 5, 6, 6.5, 7, 7.5, and 8% w/v) were evaluated in MRS broth. Acid production from carbohydrates was determined following the methods reported by Tanasupawat et al. (2002). The lactic acid isomer was analysed using the enzymatic method described by Okada et al. (1978).

16S rRNA gene sequencing analysis

The initial species identification of strain CRM56-2 was carried out using the 16S rRNA gene. It was amplified following the protocol by Phuengjayaem et al. (2017) and sequenced using universal primers [27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′TACGGYTACCTTGT-TACGACTT′3)] as described by Lane (1991) on a sequencer (Macrogen, Korea). Similarities of the 16S rRNA gene of strain CRM56-2 to the database were enumerated using the Ezbiocloud web-based tools (Yoon et al. 2017). The data were deposited in the DNA Data Bank of Japan (DDBJ), Mishima, Japan. The DDBJ accession number of strain CRM56-2 was LC742934.

Genomic sequencing, assembly and features

The genomic DNA was extracted following the procedure outlined by Yamada and Komagata (1970). Subsequently, the preparation of the library and sequencing were carried out at the Faculty of Life Sciences, Tokyo University of Agriculture, utilising the Nextera DNA Flex Library Prep Kit and the Illumina MiSeq platform with MiSeq v3 reagent kit (600 cycles). The genomic quality was determined using FastQC web-based tool, and TrimGalore web-based tool was applied to remove adaptors and low-quality reads. Filtered Illumina reads were assembled using Unicycler (Galaxy Version 0.4.8.0), and CheckM was used to evaluate the genomic quality (Park et al. 2015). The JSpeciesWS online server tool (Richter & Rosselló-Móra 2009; Richter et al. 2016) and Genome-to-Genome Distance Calculator (GGDC 2.1) (Meier-Kolthoff et al. 2013) were used to examine the average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) data. TYGS web server (https://tygs.dsmz.de/) was used to construct the phylogenomic tree (Meier-Kolthoff & Göker 2019). Furthermore, the circular genomic map was generated using the Proksee Server (Stothard et al. 2019).

Gene annotation and functional prediction

The DFAST server (Tanizawa et al. 2018), Rapid Annotation Server Technology (RAST) (Aziz et al. 2008), PATRIC (Davis et al. 2020), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016) were utilised to annotate the draft genome. PlasmidFinder (Carattoli et al. 2014) was used to detect plasmid. Genomic features are listed in Table 1. ResFinder (Bortolaia et al. 2020) was utilised to investigate antibiotic resistance genes. PathogenFinder was applied to predict pathogenicity (Cosentino et al. 2013). Putative prophage sequences were annotated and determined using the PHAge Search Tool Enhanced Release (PHASTER) (Arndt et al. 2016). The dbCAN meta server (https://bcb.unl.edu/dbCAN2/blast.php) with HMMER: biosequence analysis with profile hidden Markov models (version: 3.3.2) was used to identify carbohydrate-active enzymes, and all data produced by dbCAN depended on the CAZy database’s family classification (http://www.cazy.org/) (Cantarel et al. 2009; Zhang et al. 2018). The pathways and genes of strain CRM56-2 were examined and annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Kanehisa et al. 2016).

In vitro Probiotic Assays

Cell suspension preparation

Strain CRM56-2 was cultured in MRS broth for 24 h at 30°C. Subsequently, the cell-free supernatant was removed by centrifugation at 14,000 rpm for 10 min at 4°C. The cells were washed with phosphate buffer (0.1 M, pH 7.2) and resuspended in phosphate buffer (0.1 M, pH 7.2) to obtain a cell suspension of 109 CFU/mL.

BSH activity

The Kingkaew et al. (2022) approach was utilised to assess BSH activity. Taurodeoxycholic acid sodium salt (TDCA) [0.5% (w/v)] and calcium chloride (CaCl2) [0.037% (w/v)] were added to MRS agar medium. A CRM56-2 cell suspension was spotted onto the agar, and the plates underwent a 72 h anaerobic incubation period at 37°C. Halos or opaque white colonies with halos around them suggested BSH activity. The control was MRS agar containing no TDCA and CaCl2.

Assimilation of cholesterol

Using MRS broth supplemented with cholesterol-polyethylene glycol (PEG) 600 (Sigma, India) at a final concentration of 100 μg/mL, the capacity of strain CRM56-2 to absorb cholesterol was assessed. The suspension of strain CRM56-2 (1%, v/v) was added into the MRS broth containing cholesterol-PEG 600 and incubated at 37°C for 24 h under anaerobic conditions. Following the procedure of Tomaro-Duchesneau et al. (2014), the cholesterol was isolated, and the concentration of cholesterol was quantified using the technique described by Rudel and Morris (1973). The cholesterol concentration was compared to a reference curve prepared using a cholesterol stock solution. The ability to assimilate cholesterol was determined by calculating the percentage of assimilated cholesterol (%) at each incubation, as follows:

(1) Assimilated cholesterol (μg/mL)=[Cholesterol (μg/mL)(0 h)-Cholesterol (μg/mL)(24 h)]

(2) Assimilated cholesterol (%)=Assimilated cholesterol (μg/mL)Cholesterol (μg/mL)(0 h)×100

Acid and bile tolerance properties

The acid and bile tolerance properties of strain CRM56-2 were determined according to the method of Hyronimus et al. (2000). For acid tolerance, 2% of LAB suspension (109 CFU/mL) was inoculated into MRS broth with a pH of 3.0 and 6.5, and incubated anaerobically at 37°C for 180 min. The samples were collected at 0 min and 180 min of incubation time to enumerate viable cells. For bile salt tolerance, 2% of LAB suspension (109 CFU/mL) was inoculated into MRS broth with a pH of 8.0, containing bile salt (0.3% (w/v)), as well as without supplementation of bile salt, and incubated anaerobically at 37°C for 3 h. The samples were collected at 0 min and 180 min of incubation time to count the remaining viable cells. The results were described as colony-forming units per milliliter (CFU/mL).

The ability of adhesion to Caco-2 cells

The adhesion ability of strain CRM56-2 to adhere to the intestinal epithelium was evaluated following the method reported by Bustos et al. (2012). Briefly, Caco-2 cells were inoculated into 24-well tissue culture plates at a concentration of 5 × 105 cells/mL and incubated at 37°C with 5% CO2 for 15 days, with culture medium changes every 72 h. Overnight cultures of strain CRM56-2 in MRS broth were collected by centrifugation at 14,000 rpm for 10 min at 4°C and cleansed with phosphate buffer solution. Subsequently, the strain CRM56-2 cells (109 CFU/mL) were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and inoculated onto the Caco-2 cells in each well, followed by incubation at 37°C with 5% CO2 for 90 min. The cells were washed thrice with PBS and lysed with 0.05% (v/v) Triton-X solution. The released bacterial cells were serially diluted, spotted onto MRS agar, and incubated at 37°C for 2 days. The adhesive capability was expressed as the percentage of adhesive cells to Caco-2 cells to the total sum of bacteria (CFU/mL). Lacticaseibacillus rhamnosus GG was used as a positive control.

RESULTS AND DISCUSSION

Strain Identification

Strain CRM56-2 is a Gram-positive, catalase-negative, facultatively anaerobic rod. It synthesises DL-lactic acid from D-glucose homofermentative and does not produce gas from glucose. The strain can grow at a temperature range of 15°C–45°C, a pH range of 2–9, as well as in the presence of NaCl (1%–6% (w/v)). Strain CRM56-2 cannot hydrolyse arginine or reduce nitrate and does not form slime. Acid is generated from various sugars such as D-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, D-mannose, D-maltose, D-mannitol, D-melibiose, D-raffinose, L-rhamnose, D-ribose, salicin, D-sorbitol, D-saccharose, D-trehalose and D-xylose. Using the entire sequences of the 16S rRNA gene(1,567 bp), strain CRM56-2 was closely related to L. paraplantarum DSM 10667T,L.pentosus DSM 20314T, L. plantarum ATCC 14917T and L. argentoratensis DSM16365T, with a similarity of 99.73%, 99.93%, 100% and 100%, respectively.

Based on the phylogenomic tree (Fig. 1), strain CRM56-2 was grouped with various strains of L. plantarum. A dDDH score of 98.3% was observed between strain CRM56-2 and L. plantarum DSM ATCC 14917T and/or 20174T, which was the highest dDDH value among closely related species (Table 2). Furthermore, this strain exhibited the highest ANIb and ANIm values of 98.92% and 99.27%, respectively, to L. plantarum ATCC 14917T (Table 2). The ANI and dDDH values, which were higher than the species boundary value (ANI > 95%–96%), confirmed that strain CRM56-2 was unequivocally identified as L. plantarum (Chun et al. 2018). Therefore, conventional tests and genomic investigations confirm that strain CRM56-2 belongs to L. plantarum.

Genomic Features of Strain CRM56-2

Based on Table 1, L. plantarum CRM56-2 (JAEMUU00000000) had a genome size of 3,373,611 bp with an N50 of 216,722, L50 of 5, and a genome coverage of 437×. The DNA G+C content of CRM56-2 was 44.3%. The genome sizes and G+C content fell within the 3 Mb–3.6 Mb range and 44%–45%, respectively. These values are consistent with previous reports for this species (Martino et al. 2016; Surve et al. 2022). This finding represents the inaugural report of L. plantarum isolation from a fermented plant source in northern Thailand. PGAP annotation reported 3,270 genes, including 3,079 coding genes, 111 pseudogenes, 80 RNA genes, 71 tRNAs and 4 ncRNAs. The genomic statistics are shown in Table 3. Strain CRM56-2 lacked CRISPRs and Supplementary Material Fig. 1 illustrates its subsystems. Fig. 2 shows the circular genome of strain CRM56-2.

CONCEIVABLE GENE FACTORS IN PROTEASE ACTIVITIES, METABOLISM OF CARBOHYDRATES AND BENEFICIAL METABOLITES OF L. PLANTARUM CRM56-2

Enzymes-associated genes with conserved proteolytic and metabolic sugar systems could be found in strain CRM56-2, allowing for the performance of a functional genomic investigation. The genome of strain CRM56-2 encodes several proteases, such as peptidases (pep), proteinase (prt), and an oligopeptide ABC transport system (opp). Peptidase enzymes cleave various compounds, including asparagine, casein, cysteine, glutamate-derived peptides, leucine, methionine, proline and serine (De Jesus et al. 2022) (Refer Supplementary Material Table S1 for details).

Various enzymes related to the breakdown and utilisation of carbohydrates, such as chitobiose, fructose, galactose, glucose, mannose and sucrose, were identified in the genome of L. plantarum CRM56-2. These include 6-phospho-beta-glucosidase, glucokinase, phosphoglucomutase and phosphomannose isomerase. Moreover, genes involved in transporting cellobiose, fructose, glucose and mannose, mainly through the PTS system, were also discovered (De Jesus et al. 2022) (Refer Supplementary Material Table S2 for details).

Furthermore, the analysis of carbohydrate-active enzymes (CAZymes) genes in the L. plantarum CRM56-2 genome revealed that they belong to carbohydrate-binding modules (CBMs) (CBM32, CBM34, CBM48) (n = 3), glycoside hydrolases (GHs) (GH1, GH2, GH13, GH25, GH31, GH32, GH36, GH38, GH42, GH65, GH70, GH73, GH78, GH85, GH126, GH170) (n = 48), and glycosyltransferases (GTs) families (GT2, GT4, GT5, GT26, GT28, GT32, GT35, GT51) (n = 31) (see Supplementary Material Table S4 for details). The high number and diversity of CAZyme genes suggests that L. plantarum CRM56-2 can utilise a variety of monosaccharides and polysaccharides as energy sources and synthesise molecules. Notably, prebiotics associated with human gut health and found in oligosaccharides are degraded by GH13 and GH32. The GH families also play a key role in oligosaccharide synthesis, indicating that strain CRM56-2 and other probiotics may be used as prebiotics (Abriouel et al. 2017). In addition, GTs expedite the transfer of sugars from activated donor molecules to certain acceptors, they are necessary to produce structural surfaces that the host immune system is able to recognise (Mazmanian et al. 2008).

In summary, the abundance and diversity of CAZymes genes in L. plantarum CRM56-2 suggest that this strain has a strong potential for immunomodulation and pathogen prevention as a probiotic.

Several genes encoding essential enzymes involved in the fermentation process were identified in L. plantarum CRM56-2, including acetate kinase, glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate isomerase, glucokinase, phosphoglycerate kinase, phosphoketolase, pyruvate kinase, pentose-5-phosphate 3-epimerase, lactic acid dehydrogenase and others (De Jesus et al. 2022). These enzymes play a critical role in the production of acetate or lactate (De Jesus et al. 2022). The study also investigated genes associated with vitamin production, such as dihydrofolate reductase (B9), riboflavin kinase (B2), and thiamine pyrophosphokinase (B1), as well as butyrate-associated genes (Supplementary Material Table S3 for details) (Botta et al. 2017). KEGG annotation revealed butanoate metabolism in L. plantarum CRM56-2, with genes implicated in butyric acid synthesis linked to the complimentary functions of a medium-chain thioesterase and FASII in CRM56-2.

These metabolic characteristics help the CRM56-2 strain ferment substances and produce useful metabolites such bioactive peptides, lactate, SCFA and vitamins. Key enzymes include acetate kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, phosphoketolase, peptidases, proteinases, pyruvate kinase, thiamine pyrophosphokinase and riboflavin kinase are associated with the Embden-Meyerhof (EMP) or phosphoketolase pathways and proteolysis in strain CRM56-2. Moreover, the study investigated butyric acid production-associated genes linked to the complementary activities of the FASII pathway and the medium-chain acyl-ACP thioesterase. Based on these findings, the presence of these metabolic-associated genes was similar to the other L. plantarum strains (Botta et al. 2017). These findings could have illustrated a previously unexplained metabolic pathway for the production of butyric acid in L. plantarum. Furthermore, these beneficial bioproducts are helpful in GIT inflammation.

Cholesterol-Lowering Activities

This study assessed the BSH activity BSH and cholesterol assimilation ability of strain CRM56-2 to evaluate its hypocholesterolemic effects (Table 4).

BSH and Cholesterol Assimilation

This study examined the BSH activity BSH and cholesterol assimilation ability of strain CRM56-2 to assess its hypocholesterolemic effects. The strain exhibited BSH activity, confirmed by the choloylglycine hydrolase (bsh) gene, and demonstrated the ability to assimilate cholesterol at 75.94%. These properties indicate the presence of cholesterol-lowering effects on the host, making BSH activity a desirable probiotic characteristic according to the FAO/WHO Guidelines for the Evaluation of Probiotics in Food (FAO/WHO 2002). However, deconjugated bile due to BSH activity may pose safety concerns, as it can negatively impact lipid digestion, disrupt intestinal environments, lead to gallstones, and be converted to carcinogens. Fortunately, CRM56-2 lacked genes to generate secondary bile salts, indicating no safety concerns related to hazardous secondary bile compounds.

The genome analysis of strain CRM56-2 showed cholesterol assimilation-associated genetic elements, including fba, ccpA and glgP. The capacity of the strain to absorb cholesterol is explained by the genes linked with cholesterol assimilation, which encode membrane-related proteins that can bind to the cholesterol molecule and further integrate it into the cell. LAB may absorb cholesterol from the GIT by attaching to the surface and utilising putative enzymatic activities, affecting the process of cholesterol absorption (Kingkaew et al. 2023). The cholesterol-lowering effects-associated genes are summarised in Table 5. In conclusion, strain CRM56-2’s capacity to deconjugate bile salts and assimilate cholesterol makes it a promising probiotic candidate with potential hypocholesterolemic effects on the host.

In vitro Probiotic Properties

Acid and bile tolerance and adhesion ability

For the assessment of acid tolerance, strain CRM56-2 was inoculated in MRS broth with a pH of 3.0 and incubated for 180 min. The viability of the strain was reduced from 2.4 × 107 to 5.8 × 106 CFU/mL. To evaluate bile tolerance, strain CRM56-2 was incubated in MRS broth supplemented with 0.3% bile salt at pH 8.0 for 3 h. The viability of the strain slightly increased from 2.7 × 106 to 7.7 × 106 CFU/mL (Table 3). Furthermore, the adhesion ability of strain CRM56-2 and L.rhamnosus GG was found to be approximately 0.40 ± 0.15% and 0.40 ± 0.05%, respectively.

Probiogenomic Characteristics and Safety Assessment

Probiogenomic characteristics

The genome of L. plantarum CRM56-2 contains encoded genes associated with stress response in the GIT and adhesion ability, including ATP-dependent ClpX protease, chaperones (GroeL, DnaJ, DnaK), enolase, F0F1 ATP system genes, Na+/H+ antiporter NhaC, glycine/betaine ABC transporter permease, S-ribosylhomocysteine lyase, two-component sensor histidine kinase, ornithine decarboxylase, serine protease HtrA, and others, as shown in Table 6. Probiotictraits are strain-specific, motivating searching for new superior strains. L. plantarum CRM56-2 showed excellent tolerance to acid and bile salt conditions. Additionally, analysis of the strain’s genome indicated that its proteinaceous compounds play a role in environmental and genetic processing information and metabolic function, suggesting the relevance of these genes in maintaining the biological function of strain CRM56-2 in specific contexts or hosts. The strain’s core genome contained genes involved in stress response mechanisms, such as bile efflux and proton extrusions, metabolic response, heat shock/chaperones protein synthesis expression and transcriptional regulators. These genetic components may be crucial to the bacterial endurance of strain CRM56-2 in the human GIT. Probiotic activity requires adhesion to the intestinal mucosa epithelium, and microbial surface proteins have been linked to colonisation (Ye et al. 2020). Genes encoding cell-surface proteins, such as elongation factor Tu and lipoprotein signal peptide, were found in L. plantarum CRM56-2. The strain also contained a gene encoding LPXTG-specific sortase, relevant to strain adherence to surrounding epithelial tissue. Cell-surface proteins known as sortase-dependent proteins are crucial for adhesion (Alayande et al. 2020). The adhesion-associated genes discovered in strain CRM56-2 may thus improve strain stability and aid in successful colonisation.

Safety assessment (pathogenicity, antibiotic resistance genes and mobile genetic elements)

After a comprehensive genome analysis, the strain CRM56-2 was identified and predicted to be a non-pathogenic microorganism (Table 7). The genome lacked mobile genetic elements such as plasmids and ARGs (Tables 1 and 7). Additionally, six prophage regions, including two intact prophages ranging in size from 29.5 to 44 Kb, three incomplete prophages (5.4 to 23.3 Kb), and one questionable prophage (18.8 Kb), were integrated into the chromosome (Supplementary Material Table S5 for details). Although virulent genetic elements were present, including the capsular polysaccharide biosynthesis protein (cps4E) and exopolysaccharides biosynthesis proteins (cps4B and cps2B), the genome of the strain CRM56-2 contained no ARGs. It was identified as a non-human pathogen, indicating that the strain is safe and minimises the chance of spreading ARGs to the host’s gut normal flora.

Phages are frequently found in the genomes of Lactobacillus species (probiotic), but no virulence factors or genes associated with pathogenic qualities were found in these phage regions. Prophages may improve bacterial fitness in unfavourable environments (Pei et al. 2021). Capsular- and exo-polysaccharide genes associated with virulence elements were explored. Exopolysaccharides improve the capacity of bacteria to survive under oxidative and osmotic stress and contribute to its ability to adhere (Diale et al. 2021). Additionally, commercial strains like the accepted Generally Recognised as Safe (GRAS) L. plantarum 299V contained the hemolysin III gene (hlyIII), as well as a number of Lactobacillus strains. If no additional virulence genes have been found in the genome, strains with the hlyIII gene are often regarded as benign. Numerous studies have been conducted on hemolysin III and its harmlessness in lactobacilli. Therefore, this proteinaceous compound is not a serious concern (Surachat et al. 2017). Because they increase bacterial survival, these genes are advantageous to the bacterium and may be required when viable cells are needed.

CONCLUSION

In this study, L. plantarum CRM56-2 was isolated from Camellia sinensis and found to express BSH activity, which was indicated by the development of an opaque white colony. Additionally, CRM56-2 could withstand acidic and bile salt environments, metabolise cholesterol by over 70%, and possibly attach to Caco-2 cells. The genomic assessment of L. plantarum CRM56-2 highlighted its appeal as a promising probiotic. The strain was deemed harmless due to the absence of ARGs, plasmids, and virulent genetic features. Furthermore, the strain contains several genes involved in the tolerance of acid and bile salts, adhesive ability, and other beneficial impacts. Based on the in vitro and in silico investigations, it was concluded that strain CRM56-2 has health-promoting benefits and probiotic features, making it a promising probiotic. The genetic information of this strain supported its favorable traits.

Supplementary Information

ACKNOWLEDGEMENTS

We thank to Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University to Engkarat Kingkaew, and also thank to the Pharmaceutical Research Instrument Centre, Faculty of Pharmaceutical Sciences, Chulalongkorn University for providing research facilities. This research is funded by Thailand Science research and Innovation Fund Chulalongkorn University (HEA663300001).

Figure 1 The phylogenomic tree was generated using whole genome sequencing data from strain CRM56-2 and closely related type strains.

Figure 2 Circular genomic map of L. plantarum CRM56-2. The information is indicated as follows: Open reading frames (ORFs) (purple), GC skew (+) (blue), GC skew (–) (yellow) and GC content (pink).

Table 1 Genomic features of Lactiplantibacillus plantarum CRM56-2 and L. plantarum 299V.

Attribute(s)	CRM56-2	299V	
Source	Fermented tea leaves	Healthy human intestinal mucosa	
Accession no.	JAEMUU000000000F	LEAV00000000F	
Genome size (bp)	3,373,611c	3,302,055c	
Plasmids	0E	2 (rep28, 98.17% identity; rep38, 99.0% identity)E	
Genome qualities:	
- Genome quality	Gooda	Gooda	
- Completeness (%)	100b	99.35b	
- Coarse consistency	98.2a	98a	
- Fine consistency	95.6a	96.2a	
G+C content (%)	44.3c	44.4c	
Genome coverage	437xf	48xf	
N50	216,722c	173,004c	
L50	5c	8c	
No. of contig	204c	67c	
No. of subsystem	235c	232c	
No. of coding	3,449c	3,264c	
sequences			
No. of RNA	73c	60c	
No. of CRISPRS	0D	0D	
Notes:

a = Data obtained from PATRIC;

b = Data obtained from CheckM;

c = Data obtained from RAST web-based tool;

D = Data obtained from DFAST annotation;

E = Data obtained from PlasmidFinder;

F = Data obtained from NCBI.

Table 2 ANIb and ANIm (%) and the digital DNA-DNA hybridisation (dDDH) values between the draft genomes of the strain CRM56-2; L. plantarum DSM ATCC and/or 14917T 20174T; L. argentorensis DSM 16365T; L. paraplantarum DSM 10667T and L. pentosus DSM 20314T.

Query genome	Reference genome	ANIb	ANIm	% dDDH (Formular 2*)	Model C.I. (%)	Distance	Prob. DDH >= 70%	G+C difference	
1	2	98.92	99.27	98.3	97.5–98.8	0.0027	97.91	0.20	
1	3	98.77	99.16	63.0	60.1–65.8	0.0465	61.85	0.74	
1	4	94.78	95.66	32.0	29.6–34.5	0.1320	0.23	0.58	
1	5	85.31	88.33	24.3	22.0–26.8	0.1793	0.01	2.03	
Notes:

* Recommended formula (identities/HSP length), which is liberated of genome length and is thus prosperous against the use of draft genome.

Table 3 Genomic statistics of strain CRM56-2.

Attribute(s)	Value(s)	
Scaffold	5	
Scaffold	234,622	
Scaffold	16	
Scaffold	55,160	
Scaffold length max	623,652	
Scaffold length min	203	
Scaffold length mean	16,537	
Scaffold length median	317	
Scaffold length standard deviation	66,461	
Scaffold number A	942,533	
Scaffold number T	937,131	
Attribute(s)	Value(s)	
Scaffold number C	733,213	
Scaffold number G	760,421	
Scaffold number N	313	
Scaffold number bp	3,373,611	
Scaffold number bp not N	3,373,298	
Scaffold number sequence	204	
Scaffold GC content overall	44.27	
Contig L50	5	
Contig N50	216,722	
Contig L90	17	
Contig N90	55,160	
Contig length max	623,652	
Contig length min	162	
Contig length mean	15,689	
Contig length median	316	
Contig length standard deviation	63,630	
Contig number bp	3,373,298	
Contig number sequence	215	
Number of gaps	11	

Table 4 In vitro probiotic properties and cholesterol-lowering activities of strain CRM56-2.

Cholesterol-lowering activities and in vitro probiotic properties	
	
	L. plantarum CRM56-2	
Cholesterol-lowering effects:		
• Bile salt hydrolase (BSH)	+	
• Cholesterol assimilation (%)	75.94	
	
- Simulated gastric phase (pH 3)		
• 0 h	2.4 × 107	
• 3 h	5.8 × 106	
	
- Simulated intestinal phase (pH 8.0, 0.3% bile salt)		
• 0 h	2.7 × 106	
• 3 h	7.7 × 106	
	
Adhesion ability (%)	0.40 ± 0.15	

Table 5 Cholesterol-lowering genes (Deconjugation of bile salt and cholesterol assimilation).

Gene	Gene description	Length (bp)	
For deconjugation of bile salt	
bsh	Choloylglycine hydrolase	975	
For cholesterol assimilation abilities	
ccpA	Catabolite control protein A	978	
fba	Class II fructose-1,6-bisphosphate aldolase	864	
glgP	Glycogen phosphorylase	2,403	
-	FMN-binding protein	369	
Note: Data obtained from DFast annotation.

Table 6 Predicted proteins identified in the genome of L. plantarum CRM56-2 strain involved in acid and bile tolerance and adhesion/interaction.

Putative function	Genes	Predictive protein	Length (bp)	
Adhesion or interaction with the host	srtA	Class A sortase	705	
dltD	D-Alanyl-lipoteichoic acid biosynthesis protein DltD	1,278	
dltA	D-Alanylation of LTA	1,527	
glnH1	Glutamine ABC transporter substrate- binding protein	837	
lspA	Lipoprotein signal peptidase	450	
tuf	Elongation factor Tu	1,188	
mtsA	Manganese ABC transporter substrate- binding protein	894	
eno2	Enolase 2	1,329	
gapB	Type I glyceraldehyde-3-phosphate dehydrogenase	1,002	
groS	Co-chaperonin GroES	285	
groL	Chaperonin GroEL	1,626	
glnA	Glutamine synthase	1,347	
pgi	Glucose-6-isomerase	1,353	
	
Acid stress	atpC	ATP synthase subunit epsilon	429	
atpD	ATP synthase subunit beta	1,404	
atpA	ATP synthase subunit alpha	1,521	
atpG	ATP synthase subunit gamma	945	
atpH	ATP synthase subunit delta	546	
atpF	ATP synthase subunit B	516	
atpB	ATP synthase subunit A	714	
atpE	ATP synthase subunit C	213	
recA	Protein RecA (recombinase A)	1,143	
relA	GTP pyrophosphokinase	2,268	
groS	Co-chaperonin GroES	285	
groL	Chaperonin GroEL	1,626	
htrA	Serine protease	1,263	
aspS	Aspartate-tRNA ligase	1,797	
	
Acid stress/Bile resistance	gpmA1	2,3-Bisphosphoglycerate dependent phosphoglycerate mutase 1	639	
gpmA2	2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase 2	693	
dnaK	Chaperone protein DnaK	1,869	
dnaJ	Chaperone protein DnaJ	1,143	
glmU	Bifunctional UDP-N acetylglucosamine diphosphorylase/glucosamine phosphate	1,383	
luxS	S-Ribosylhomocysteine lyase	477	
gadB	Glutamate decarboxylase; GABA transporter	1,410	
nha1	Na+/H+ antiporter NhaC	1,425	
nha2	Na+/H+ antiporter NhaC	1,401	
clpX	ATP-dependent Clp protease ATP-binding subunit clpX	1,266	
	
Bile resistance	nagB	Glucosamine-6-phosphate deaminase	714	
pyrG	CTP synthase	1,614	
argS	Arginine-tRNA ligase	1,689	
rpsC	30S Ribosomal protein S3	654	
rpsE	30S Ribosomal protein S5	501	
rplD	50S Ribosomal protein L4	624	
rplE	50S Ribosomal protein L5	543	
rplF	50S Ribosomal protein L6	537	
Note: Data obtained DFast annotation.

Table 7 Pathogenicity prediction, prophage detection and antibiotic resistance genes (ARGs) analysis from PathogenFinder and ResFinder of CGE and PHASTER (Default program settings applied).

Attribute/Strain	L. plantarum CRM56-2	L. plantarum 299v	
Probability of being a human pathogen	0.211	0.185	
Input proteome coverage (%)	0.82	0.48	
Matched pathogenic families	0	0	
Matched not pathogenic families	26	15	
Conclusion	Non-human pathogen	Non-human pathogen	
No. of phage regions	6	4	
ResFinder	No resistance	No resistance	

AUTHORS’ CONTRIBUTIONS: Engkarat Kingkaew: Investigation, methodology, data curation, writing – review and editing.

Naoto Tanaka: Methodology, writing – review and editing.

Yuh Shiwa: Investigation, methodology, data curation.

Jaruwan Sitdhipol: Methodology, funding acquisition.

Rattanatda Nuhwa: Investigation, methodology, data curation, writing.

Somboon Tanasupawat: Supervision, funding acquisition, writing – review and editing.
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