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Microbiology and Biotechnology Letters

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Food Microbiology  |  Probiotics in Nutrition and Health

Microbiol. Biotechnol. Lett. 2023; 51(4): 403-415

https://doi.org/10.48022/mbl.2308.08008

Received: August 21, 2023; Revised: September 25, 2023; Accepted: October 6, 2023

Characterization of L-(+)-Lactic Acid Producing Weizmannia coagulans Strains from Tree Barks and Probiogenomic Evaluation of BKMTCR2-2

Jenjuiree Mahittikon1, Sitanan Thitiprasert2, Nuttha Thongchul2, Naoto Tanaka3, Yuh Shiwa3,4, Nitcha Chamroensaksri5, and Somboon Tanasupawat1*

1Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2Center of Excellence in Bioconversion and Bioseparation for Platform Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand
3Department of Molecular Microbiology, Faculty of Life Sciences, Tokyo University of Agriculture, Tokyo 156-8502, Japan
4NODAI Genome Research Center, Tokyo University of Agriculture, Tokyo 156-8502, Japan
5National Biobank of Thailand (NBT), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand

Correspondence to :
Somboon Tanasupawat,        Somboon.T@Chula.ac.th

Abstract

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This study aimed to isolate and identify L-(+)-lactic acid-producing bacteria from tree barks collected in Thailand and evaluate the potential strain as probiotics. Twelve strains were isolated and characterized phenotypically and genotypically. The strains exhibited a rod-shaped morphology, high-temperature tolerance, and the ability to ferment different sugars into lactic acid. Based on 16S rRNA gene analysis, all strains were identified as belonging to Weizmannia coagulans. Among the isolated strains, BKMTCR2-2 demonstrated exceptional lactic acid production, with 96.41% optical purity, 2.33 g/l of lactic acid production, 1.44 g/g of lactic acid yield (per gram of glucose consumption), and 0.0049 g/l/h of lactic acid productivity. This strain also displayed a wide range of pH tolerance, suggesting suitability for the human gastrointestinal tract and potential probiotic applications. The whole-genome sequence of BKMTCR2-2 was assembled using a hybridization approach that combined long and short reads. The genomic analysis confirmed its identification as W. coagulans and safety assessments revealed its non-pathogenic attribute compared to type strains and commercial probiotic strains. Furthermore, this strain exhibited resilience to acidic and bile conditions, along with the presence of potential probiotic-related genes and metabolic capabilities. These findings suggest that BKMTCR2-2 holds promise as a safe and effective probiotic strain with significant lactic acid production capabilities.

Keywords: Genomic analysis, lactic acid bacteria, L-(+)-lactic acid, tree bark, probiogenomic, Weizmannia coagulans

Graphical Abstract


Introduction

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Weizmannia coagulans has garnered significant attention as a promising strain due to its probiotic attributes and remarkable resilience in harsh conditions. This Gram-positive bacterium belongs to a rod-shaped, sporeforming genus known for its capacity to produce lactic acid [1]. Most studies on W. coagulans have focused on strains isolated from animal sources, such as the gastrointestinal tracts of mammals or fermented foods. However, recent research suggests that bacteria isolated from plant sources exhibit unique characteristics and higher resilience to environmental stressors [2]. Therefore, exploring the potential of W. coagulans strains isolated from tree bark could provide valuable insights into their adaptability and probiotic attributes.

Numerous studies support the safe utilization of probiotic strains belonging to W. coagulans for managing significant depression accompanied by irritable bowel syndrome, effectively alleviating associated clinical symptoms such as vomiting, diarrhea, and irregular bowel movements [3]. Furthermore, these strains have shown the potential to improve conditions such as bloating and abdominal pain by promoting a healthy microbiome within the human gastrointestinal tract [4]. It highlights the therapeutic benefits and potential applications of W. coagulans probiotics in the context of IBS and associated psychological disorders [5]. Based on the evidence, W. coagulans has gained considerable attention for commercial probiotic strains and is utilized in various health supplements, including LactoSpore®, SIBNO™, GanedenBC30, and PROBC. In addition, these strains have obtained Generally Recognized as Safe (GRAS) status and have been granted Qualified Presumptions of Safety (QPS) according to guidelines set forth by regulatory bodies such as the FDA and EFSA. This recognition further establishes the safety and reliability of W. coagulans strains for consumption [1].

To establish their efficacy as probiotics, it is crucial for strains commonly found in food and health supplements to withstand the challenges posed by the gastrointestinal tract, including acidic gastric acid, bile, and pancreatin. W. coagulans, with its extracellular enzymes, displays the capacity to break down complex biomolecules into smaller subunits. Additionally, successful probiotic strains must exhibit adherence to intestinal cells and antagonistic activity against pathogenic bacteria. Genomic analysis of W. coagulans determined that the transferability of antibiotic resistance genes to other bacteria was limited, and no other concerning genes were identified, signifying a favorable safety profile [1].

This study aimed to characterize W. coagulans strains obtained from various tree barks and assess their suitability as probiotics. The specific objectives were as follows: 1) isolation and identification of W. coagulans strains from tree barks; 2) evaluation of their phenotypic and genotypic characteristics; 3) screening of their lactic acid production and antimicrobial properties; and 4) conducting a comprehensive probiogenomic analysis of the most promising strain, BKMTCR2-2. The probiogenomic assessment encompassed whole-genome sequencing, safety evaluation, and examination of probiotic-associated genes.

Materials and Methods

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Isolation and identification

Tree bark was collected from Bangkok, Chaiyaphum, Nakhon Ratchasima, and Satun provinces in Thailand. Each sample was pre-treated with heat at 80℃ for 10 min. The samples were enriched in Glucose-Yeast-Peptone (GYP) medium, which consisted of 10 g of glucose, 5 g of yeast extract, 5 g of peptone, 0.25 g of KH2PO4, 0.25 g of K2HPO4, and 10 ml of salt solution. The salt solution consisted of (per 1 L) 0.4 g of MgSO4·7H2O, 0.02 g of MnSO4·5H2O, 0.02 g of FeSO4·7H2O, and 0.02 g of NaCl. The samples were incubated anaerobically at 37℃ for 48−72 h. After incubation, the isolates were streaked on GYP agar supplemented with 0.3% CaCO3 and incubated at 37℃ for three days under anaerobic conditions using AnaeroPack (Kenki), Mitsubishi Gas Chemical, Japan. Colonies surrounded by a clear zone were selected and purified until a pure culture was obtained. All strains were preserved in skim milk stock at -20℃ and stored in lyophilized ampoules [6].

Phenotypic characteristics

For phenotypic characteristics, cell shape, cell size, colonial appearance, Gram-staining, and spore formation of cells grown on GYP agar supplemented with CaCO3, incubation at 37℃ for 48 h under anaerobic conditions were observed, as described previously [7]. The biochemical and physiological characteristics, such as gas formation, catalase production, nitrate reduction, arginine hydrolysis, blood hemolysis and the effects of growth at different temperatures (15, 30, 37, 45℃), pH values (3.0, 6.0, 9.0), and NaCl concentrations (4, 6, 8% w/v), were examined. The isomer of lactic acid was enzymatically determined, as described by Tanasupawat et al. [8].

Genotypic characteristics

The 16S rRNA gene sequencing. The DNA of each strain was extracted using the colony PCR method. The 16S rRNA gene sequencing was conducted with the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') by Macrogen [9]. The 16S rRNA gene sequences were then analyzed using the Ezbiocloud web-based tools [10]. A neighbor-joining phylogenetic tree was constructed using MEGA 11 [11], and confidence values for the individual branches of the phylogenetic tree were determined through 1000 replicates [12].

Genome sequencing. Whole-genome sequencing was conducted using the Illumina MiSeq and Oxford Nanopore platforms at the Nodai Genome Research Centre, Tokyo University of Agriculture, Japan. The genomic DNA of strain BKMTCR2-2 was extracted using a NucleoBond Buffer Set III (TaKaRa, Japan) and a NucleoBond AXG20 column (TaKaRa). The short-read DNA was prepared and sequenced following the Nextera DNA Flex Library Prep Kit protocol (Illumina, USA) and the Illumina HiSeq 2000, respectively, as described in the method by Yokoyama et al. [13]. The long-read DNA sequencing was performed using Oxford Nanopore Technology. To calculate the in silico genomic identity of BKMTCR2-2 with the reference strain, the average nucleotide identity (ANIb and ANIm) and tetra-nucleotide signatures (Tetra) were determined using the JSpecies Web Server (JSpeciesWS) [14]. The Genome-to-Genome Distance Calculator (GGDC 2.1) was also used to calculate digital DNA-DNA hybridization values [15].

The 16S rRNA gene and whole-genome sequences of BKMTCR2-2 are available at the DNA Data Bank of Japan (DDBJ) and the National Center for Biotechnology Information (NCBI) under accession numbers LC769116 and JASUZX000000000, respectively. The accession numbers for W. coagulans ATCC 7050T is PRJNA238056.

Genome assembly, annotation, functional analysis, and safety evaluation

The whole-genome sequence of representative isolate BKMTCR2-2 was assembled by hybridizing reads from the short-read Illumina and the long-read Oxford Nanopore Technologies instruments [16]. The raw genome sequences were trimmed and assembled using the Unicycler (version Galaxy Version 0.5.0+galaxy1) [17, 18]. The genomic circular was generated by Proksee [19].

The gene annotation and prediction, feature analysis, and plasmid observation were carried out using DDBJ Fast Annotation and Submission Tool (DFAST) server [20], Rapid Annotation Server Technology (RAST) [21], PATRIC [22], the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [23], PlasmidFinder [24], BAGLE4 [25]. The dbCAN server was used to annotate and analyze carbohydrate-active enzyme genes (https://bcb.unl.edu/dbCAN2/blast.php) with HMMER (version: 3.3.2). The CAZy database (http://www.cazy.org/) was used to classified and generated enzyme family [26, 27]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database determined the bacterial enzymatic and genetic metabolic pathways [28]. The potential associated genes of probiotic characteristics were evaluated [18].

For the utilization of BKMTCR2-2 as a probiotic, a safety assessment was conducted based on several criteria. In this study, three probiotic reference strains, W. coagulans ATCC 7050T (NZ_CP009709), W. coagulans IS-2 (JZDH00000000) and W. coagulans GBI-30 (JPSK00000000), were compared.

The Center for Genomic Epidemiology (CGE) was used to evaluate the pathogenicity [29], antibiotic resistance gene [30], acquired virulence genes [31], and plasmids [32]. The Comprehensive Antibiotic Resistance Database, CARD, detected antibiotic resistance genes [33]. Additionally, the origin of transfer in DNA sequence was investigated by oriTfinder, a web-based tool [34].

Qualitative and quantitative analysis of screening lactic acid production

The lactic acid isomer was screened using high-performance liquid chromatography (HPLC). LAB was cultivated in GYP broth at 37℃ for 48 h, and the supernatant was collected by centrifugation. The concentration, optical purity, and other by-products were evaluated using HPLC [6].

The product yield (Yp/s), productivity, and optical purity were calculated following the methods described by Thitiprasert et al. [35]. The optical purity of lactic acid was calculated as follows:

The optical purity of lactic acid =(D(-)lactic acid - L(+)lactic acid)(D(-)lactic acid + L(+)lactic acid)×100

All experiments were done in triplicate, and results were reported as the mean ± standard deviation. Results were statistically analyzed by ANOVA (Analysis of variance) with Duncan's new multiple range test for mean comparison by SPSS 22.0 software.

Screening of antimicrobial activities

All isolates were cultivated and incubated in GYP broth at 30℃ for 48 h. The supernatants were collected by centrifugation at 14,000 rpm for 5 min. The cell-free supernatants (CFSs) were neutralized to pH 6.0 using 1 M NaOH and then boiled at 100℃ for 5 min. The antimicrobial activity was evaluated using the spot-on-lawn assay [36]. In this study, the indicator strains used were Latilactobacillus sakei JCM 1157T, Bacillus subtilis ATCC 6051T, Listeria monocytogenes ATCC 15313T, Salmonella Typhimurium ATCC 13331T, Escherichia coli ATCC 25922, and Kocuria rhizophila ATCC 4698.

Results

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Isolation and identification

Twelve strains were isolated from various tree barks, namely Terminalia chebula Retz. (TCR), Carallia brachiata (Lour.) Merr. (CBM), Chrysophyllum cainito L. (CCL), Thespesia populnea (L.) Sol. ex Corrêa (PT), Diospyros rhodocalyx Kuarz. (DR), Sauropus androgynus Merr. (SA), Artocarpus integer (Thumb.) Merr. (AI), and Mangifera caloneura Kurz. (M). The details regarding the samples, the number of strains, and their phenotypic and genotypic characteristics are provided in Tables 1 and 2 and Fig. 1.

Table 1 . Samples, collecting places, strain number, and 16S rRNA gene sequence similarity (%) of strains compared with Weizmannia coagulans ATCC 7050T.

NO.Tree bark (Scientific name)ProvinceStrain no.Similarity (%)Length (bp)Accession no.
1Carallia brachiata (Lour.) Merr.BangkokBKMCBM-199.781,371LC769107
2Chrysophyllum cainito L.BangkokBKAP-499.721,410LC769108
BKMCCL-199.571,403LC769109
BKMCCL-299.561,379LC769110
3Terminalia chebula Retz.BangkokBKMTCR2-299.121,554LC769116
4Thespesia populnea (L.) Sol. ex CorrêaBangkokBKMPT-899.221,404LC769117
5Diospyros rhodocalyx Kuarz.ChaiyaphumCPDR-399.221,430LC769111
8Sauropus androgynus (L.) Merr.Nakhon RatchasimaNKSA-199.561,371LC769113
NKSA-499.361,405LC769114
NKSA-599.631,358LC769115
9Artocarpus integer (Thumb.) Merr.SatunSTAI-498.961,450LC769106
10Mangifera caloneura Kurz.SatunSTM-1199.631,360LC769112


Table 2 . Differential phenotypic characteristics of strains.

CharacteristicsBKMCBM-1BKAP-4BKMCCL-1BKMCCL-2BKMTCR2-2BKPT-8CPDR-3NKSA-1NKSA-4NKSA-5STAI-4STM-11
Growth at 15℃++++---+++++
Growth in pH 3.0+++++--+-+--
Growth in pH 9.0+++++++++-++
Esculin hydrolysis----+++--+--
Arginine hydrolysis-+---++-----
Nitrate reduction-+--+-------
Acid production from:
Cellobiose++--+++-+-++
Mannitol+++++--+-+--
Raffinose+---+---++++
Rhamnose+-+++--+++++
Salicin+++-+++-+-++
Xylose+++++--+++++

+, positive reaction; -, negative reaction. All strains and L. rhamnosus GG showed no hemolytic activity.

Figure 1.Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence of strains.

The various phenotypic characteristics of the isolating strains are presented in Table 2. All strains exhibited a rod-shaped morphology and did not produce gas from glucose. They could grow in a medium containing 4% NaCl at pH 6.0 and temperatures of 30℃ and 45℃, but they failed to grow in the presence of 8% NaCl. Additionally, the strains showed positive results for catalase activity. In terms of sugar utilization and acid production, they could synthesize acid from arabinose, fructose, galactose, glucose, lactose, maltose, mannose, melibiose, ribose, sucrose, and trehalose. However, they were unable to hemolyse blood and produce acid from sorbitol.

In terms of genomic characteristics, all strains exhibited a 16S rRNA gene sequence similarity ranging from 98.96% to 99.78% with W. coagulans ATCC 7050T, as shown in Table 1. The phylogenetic tree, generated using the neighbor-joining algorithm based on the 16S rRNA gene sequences of the isolated strains and reference strains, is presented in Fig. 1.

Qualitative and quantitative analysis of screening lactic acid production

Fermentation kinetics, including lactic acid concentration, the yield of lactic acid, and the productivity of the isolated strain by HPLC, are presented in Fig. 2. All strains exhibited characteristics of homofermentative lactic acid production without producing acetic acid and ethanol as by-products. Under anaerobic conditions for 48 h, with an initial glucose concentration of 10 g, they produced L-(+)-lactic acid with an optical purity ranging from 84.57% to 100%. They yielded 0.34 g/l to 2.33 g/l of lactic acid production, 0.29 g/g to 1.44 g/g of lactic acid yield (per gram of sugar consumed), and had a lactic acid production rate of 0.007 g/l/h to 0.0049 g/l/h. The strain BKMTCR2-2 presented significant differences from other strains (p-value < 0.05), producing L-(+)-lactic acid with 96.41% optical purity, 2.33 g/l of lactic acid production, 1.44 g/g of lactic acid yield (per gram of glucose consumed), and 0.0049 g/l/h of lactic acid productivity.

Figure 2.Fermentation kinetics of lactic acid produced from the strains. The different letter means a significant different between group at p-value < 0.05. The capital letters (A-K), numbers (1-8) and letters (a-h) are significant differences in lactic acid yield, productivity, and concentration, respectively, which compared with all strains.

Screening of antimicrobial activities

The screening of antimicrobial activity by using spoton-lawn assay, which selected Latilactobacillus sakei JCM 1157T, Bacillus subtilis ATCC 6051T, Listeria monocytogenes ATCC 15313T, Salmonella Typhimurium ATCC 13331T, Escherichia coli ATCC 25922, and Kocuria rhizophila ATCC 4698 as indicator bacteria. All strains showed no antimicrobial activity.

Functional probiogenomic and safety assessment

Based on the results, strain BKMTCR2-2 exhibited several attractive characteristics, including growth at pH 3.0 and 9.0 under anaerobic conditions, which are assumed to represent gastric and intestinal conditions. This strain showed no hemolysis activity compared to Lactobacillus rhamnosus GG, a commercial probiotic strain, surpassing the values of other isolated strains. Consequently, the representative strain BKMTCR2-2 was chosen for whole-genome sequencing. Table 3 presents the assembly genome statistics, genomic circular for BKMTCR2-2, and a comparison to probiotic bacteria. Phylogenomic analysis revealed that the representative strain BKMTCR2-2 grouped with W. coagulans strains, displaying the closest correlation with W. coagulans ATCC7050T, exhibiting digital DNA-DNA hybridization (dDDH), ANIb, and ANIm values of 83.5%, 97.44%, and 98.27%, respectively. Based on genomic data analysis, strain BKMTCR2-2 was identified as W. coagulans. The genome of BKMTCR2-2 was sequenced using a hybridization approach, combining short-read Illumina and long-read Oxford Nanopore Technologies. The resulting genome sequence consisted of 3,269,792 base pairs, with a genomic DNA G+C content of 47.1%. The N50 value was 2,013,348, with an L50 value of 1, and the genome coverage reached 103x. CheckM analysis indicated a genome completeness of 96% for this strain. A circular genomic is displayed in Fig. 3.

Table 3 . Genomic features of W. coagulans BKMTCR2-2, W. coagulans IS-2, and W. coagulans GBI-30.

AttributeW. coagulans BKMTCR2-2W. coagulans ATCC 7050TW. coagulans IS-2W. coagulans GBI-30
SourceTree bark (Terminalia chebula Retz.)Dairy (Evaporated milk)Human fecal contaminated soilMarketed probiotic product
Accession no.PRJNA975477ANZ_CP009709AJZDH00000000AJPSK01000000A
Genome size (bp)3,270,341G3,366,995B3,446,692B3,458,616B
Plasmids0C0C0C0C
Genome qualities:
- Genome qualityGoodDGoodDGoodDGoodD
- Completeness (%)96E95.9E99.6E99.6E
- Coarse consistency96.5D96.5D96.2D96.2D
- Fine consistency95.2D95.9D95.1D94.8D
G+C content (%)47.1B46.9B46.4B46.4B
Genome coverage103x776x282.0x840.0x
N502,013,348B1B51,676B44,706B
L501B1B20B27B
No. of contig14B1B143B224B
No. of subsystem294B297B298B297B
No. of coding sequences3,740B3888B3,994B4,061B
No. of RNA103B107B87B88B
No. of CRISPRS2F3F2F5F

A, Information obtained from NCBI; B, Information obtained from RAST web-based tool; C, Information obtained from PlasmidFinder; D, Information obtained from PATRIC; E, Information obtained from CheckM; F, Information obtained from DFAST annotation; G, Information obtained from Unicycle, Galaxy.

Figure 3.Circular genomic visualization of W. coagulans BKMTCR2-2. The graphic details included open reading frames (ORFs) as blue color, guanine-cytosine (GC) content as pink color, guanine-cytosine (GC) Skew (+) as green color, and guanine-cytosine (GC) Skew (-) as purple color.

Based on DFAST and RAST annotation, the gastrointestinal survival association and beneficially healthy gastric gene as probiotic properties are illustrated in Table 5. The CGE website predicted the safety evaluation of strain BKMTCR2-2 for elevated verification for probiotic consumption. As for the safety estimation results, this strain was defined as a non-human pathogenic bacterium with no virulence, plasmid, and antibiotic resistance genes. Additional resistance gene identification was analyzed by CARD (Table 4). Furthermore, carbohydrate-active enzyme genes are annotated and shown in Supplementary 1.

Table 4 . Pathogenicity prediction, prophage detection, and antibiotic resistance genes (ARGs) analysis from PathogenFinder of CGE (Default program settings applied) of strain BKMTCR2-2 and related W. coagulans.

Attribute/StrainW. coagulans BKMTCR2-2W. coagulans ATCC 7050TW. coagulans IS-2W. coagulans GBI-30
Probability of being a human pathogen0.4660.4560.40.4
Input proteome coverage (%)0.190.180.150.15
Matched pathogenic families2211
Matched not pathogenic families4444
ConclusionNon-human pathogenNon-human pathogenNon-human pathogenNon-human pathogen
Antibiotic resistance genes (ARGs)
CARD:
- No. of perfect hits0000
- No. of strict hits3344
- No. of loose hits000210
ResFinderNo resistanceNo resistanceNo resistanceNo resistance


Table 5 . Potential genes associated to various probiotic characteristics from W. coagulans BKMTCR2-2 genome.

Putative functionGenesGene product
Acid resistance
atpBF0F1 ATP synthase subunit A
-ATP synthase subunit B
atpEF0F1 ATP synthase subunit C
atpAF0F1 ATP synthase subunit alpha
atpDF0F1 ATP synthase subunit beta
-F0F1 ATP synthase subunit delta
atpCF0F1 ATP synthase subunit epsilon
atpGF0F1 ATP synthase subunit gamma
groLChaperonin GroEL
groESCo-chaperone GroES
recARecombinase RecA
aspSAspartate-tRNA ligase
-GTP pyrophosphokinase family protein
Acid and Bile resistance
dnaKMolecular chaperone DnaK
dnaJMolecular chaperone DnaJ
glmUBifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase GlmU
-S-ribosylhomocysteine lyase
gpmI2,3-Bisphosphoglycerate-independent phosphoglycerate mutase
Bile resistance
rplD50S Ribosomal protein L4
rplE50S Ribosomal protein L5
rplF50S Ribosomal protein L6
rpsC30S Ribosomal protein S3
rpsE30S Ribosomal protein S5
pyrGCTP synthase
argSArginine-tRNA ligase
nagB_1Glucosamine-6-phosphate deaminase
nagB_2Glucosamine-6-phosphate deaminase
Gastrointestinal adherence
groLChaperonin GroEL
groESCo-chaperone GroES
gapType I glyceraldehyde-3-phosphate dehydrogenase
lspALipoprotein signal peptidase II
glnHGlutamine ABC transporter substrate-binding protein
tufElongation factor Tu
-Glucose-6-isomerase
Maintenance of organism system
ccpACatabolite control protein A
DNA and protein protection and repair
msrAPeptide-methionine (S)-S-oxide reductase MsrA
msrBPeptide-methionine (R)-S-oxide reductase MsrB
groLChaperonin GroEL
groESCo-chaperone GroES
Regulation of immune system / Acid resistance
clpBPotential immunogenic proteins
tufElongation factor Tu
lspALipoprotein signal peptidase
Metabolic rearrangement
aldBAlpha-Acetolactate decarboxylase
Transcriptional regulator
hrcAHeat-inducible transcriptional repressor HrcA
ctsRTranscriptional regulator CtsR
Fatty acid synthesis
accCAcetyl-CoA carboxylase biotin carboxylase subunit
fabDACP S-malonyltransferase
fabFBeta-Ketoacyl-ACP synthase II
fabHBeta-Ketoacyl-ACP synthase III
fabLEnoyl-[acyl-carrier-protein] reductase FabL
Vitamin synthesis (Subsystem)
Thiamin biosynthesis (Thiamin: B1)
Riboflavin metabolism (Riboflavin: B2)
NAD and NADP cofactor biosynthesis global (Niacin: B3)
Coenzyme A biosynthesis (Pantothenate: B5)
Biotin biosynthesis (Biotin: B7)
Folate biosynthesis (Folate: B9)

Discussion

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The outstanding feature of this research consisted of bacterial characterization, screening, and identification of W. coagulans strains isolated from tree bark, as well as genomic analysis associating lactic acid fermentation and probiotic properties of BKMTCR2-2.

The bacterial proposal focused on the potential of strain BKMTCR2-2 as a lactic acid fermentation and probiotic strain, aiming to overcome various limitations in lactic acid production, gastrointestinal survival, and industrial probiotic production, such as high-temperature stress, acidic environments, rapid dehydration, and associated oxidative damage. Additionally, it has been observed that strains isolated from plants exhibit more resilience to environmental stressors when compared to strains isolated from animals [2]. Therefore, the first isolation approach involved an 80℃ pretreatment of tree bark in gathering high-stress tolerant bacterial strains. All isolated strains underwent conventional characterization for bacterial identification and attribute evaluation, including phenotypic and genotypic characteristics. They were Gram-positive, rod-shaped, catalase-negative, high-temperature-tolerating, facultatively anaerobic bacteria. Based on 16S rRNA gene sequencing and the phylogenetic tree, two branches were identified, one forming a group with W. coagulans ATCC7050T and the other with W. coagulans 2-6. All strains belonged to Weizmannia coagulans. Based on the primary screening of lactic acid production results, all strains were described as homofermentative L-(+)-lactic acid-producing bacteria. BKMTCR2-2 demonstrated outstanding phenotypic traits among the isolated strains, including a wide range of pH growth abilities. This strain also produced a high concentration, yield, and productivity of L- (+)-lactic acid, which is known to have no harmful effects on human wellness even when highly accumulated in the human body [37]. The stability of acids is a crucial quality for survival throughout the gastrointestinal tract. Therefore, the significant production of lactic acid by the selective strain serves as clear evidence that this particular strain is capable of thriving in acidic environments. In addition, the selected strain's elevated production of lactic acid has the potential to lower the pH of the surrounding environment. Consequently, this decrease in pH creates a selective environment that inhibits the pathogens that are susceptible to acidic conditions. Hence, strain BKMTCR2-2 was chosen as the representative strain for the probiotic proposal.

The whole-genome sequencing of the strain was utilized for bacterial identification, safety assessment, and prediction of probiotic-related genes. To improve the precision and quality of the bacterial genome, both longread and short-read DNA of BKMTCR2-2 were combined using whole-genome assembly methods [38]. Strain BKMTCR2-2 was classified as W. coagulans based on dDDH and ANI values, with an ANI value greater than 95−96% as the cutoff for species delineation [15, 39].

Regarding safety evaluation, strain BKMTCR2-2 was identified as a non-human pathogen. The absence of hemolysis activity compared to L. rhamnosus GG, a widely recognized commercial and research probiotic strain, was notable. Strain BKMTCR2-2 demonstrated the presence of the VanH (VanO cluster), VanT (VanG cluster), and VanY (VanM cluster) genes, which are responsible for encoding the synthesis of D-lactate, Dserine, and D,D-carboxypeptidases, respectively. The presence of resistance genes may raise concerns about probiotic safety assessment. However, the resistance genes found in this strain have putative functions related to lactic acid metabolism rather than conferring resistance to antibiotics. Furthermore, the absence of plasmids and the origin of transfer (oriT), vital components for horizontal gene transfer, in the genome of strain BKMTCR2-2 adds a layer of safety [40, 41]. The ability of probiotics to survive and colonize the gastrointestinal tract is a critical factor in determining their efficacy. Strain BKMTCR2-2 exhibited a wide range of pH growth abilities, including resilience against gastric acid and bile conditions. The previously mentioned phenotype was supported by the presence of F0F1 ATP synthase (Cluster: atpA, atpB, atpC, atpD, atpE, and atpG genes), which is responsible for pH regulation through the production of a proton-motive force [42]. Furthermore, the simultaneous expression of groEL and groES genes, which encode the co-chaperonins GroEL and GroES, respectively, has been observed to improve bacterial survival in harsh environments by promoting protein folding and supporting adherence to the intestinal epithelium [43]. The effectiveness of the co-chaperonins GroEL and GroES relates to the interactions of chaperones DnaK and DnaJ, which are encoded by dnaK and dnaJ, respectively.

Additionally, the tuf gene plays a role in epithelial adhesion and immune modulation [44]. The crucial chaperone protein, clpB, encoded by clpB, regulates the host immune system and collaborates with DnaK and DnaJ [45]. In addition, probiotics play an essential function in the alimentary tract by synthesizing beneficial molecules. Additionally, subsystems and genes are responsible for the biosynthesis and metabolism of essential constituents, including fatty acids, lipids, organic acids, amino acids, vitamins, and cofactors. The genetic elements identified in strain BKMTCR2-2 underscore its ability to enhance the nutritional status and support metabolic processes, fostering a beneficial symbiotic interaction between the probiotic and the human host.

Conclusion

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This study isolated twelve strains from various tree barks and characterized them. These strains exhibited characteristics of lactic acid production without producing harmful by-products. BKMTCR2-2 showed outstanding phenotypic traits, including wide-range pH growth abilities and high production, yield, and productivity of L-(+)-lactic acid. The taxonomic classification of BKMTCR2-2 was determined to be W. coagulans through whole-genome sequencing. The safety assessment verified that BKMTCR2-2 is a non-human pathogenic bacterium with no hemolytic activity. The strain also exhibited genes related to lactic acid metabolism rather than antibiotic resistance. BKMTCR2-2 demonstrated resilience against gastric acid and bile conditions and showed the presence of essential genes associated with bacterial survival in harsh environments. Additionally, the strain demonstrated proficiency in genes related to the biosynthesis and metabolic pathways of beneficial compounds, including fatty acids, lipids, organic acids, amino acids, vitamins, and cofactors. Furthermore, it displayed adherence genes that increase the survival rate in the human gastrointestinal tract.

The results of this study demonstrate the potential of strain BKMTCR2-2 as a safe and effective probiotic in improving the human host's nutritional status and metabolic functions. Acknowledging that these findings are based on the present data and current understanding is essential. Further research and comprehensive safety assessments, including in vitro and in vivo studies, would be beneficial to confirm the probiotic safety profile. Investigating the characteristics and probiotic capabilities of W. coagulans strains obtained from tree bark could contribute to developing novel probiotic compositions and provide valuable knowledge.

Acknowledgments

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This research was supported by the Development and Promotion of Science and Technology Talents Project (DPST), Thai government scholarship as a scholarship to Jenjuiree M. (561060), the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), Graduate School, Chulalongkorn University, and the Faculty of Pharmaceutical Sciences, Chulalongkorn University for providing research fund (Grant number Phar2565-RG002) to Dr. Somboon Tanasupawat. The genomic analysis was associated by the Department of Molecular Microbiology, Tokyo University of Agriculture. The authors thank the Pharmaceutical Research Instrument Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University for providing research facilities; Dr. Engkarat Kingkaew; Dr. Sukanya Phuengjayaem, Dr. Saranporn Poothong and all friends for consistency encouragement to pass through the research project.

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