Preliminary Characterization of LAB Isolates
The strains isolated from fermented rice underwent initial characterization through physiological and biochemical testing. A diverse array of both Gram-positive and Gram-negative strains were obtained. Subsequent antibacterial assays against S. Typhi revealed that only one strain exhibited significant activity, as evidenced by the observed zone of inhibition (Fig. 1A).
The antimicrobial activity present in the cell-free supernatant (CFS) was subjected to enzymatic treatment with various enzymes, including trypsin, chymotrypsin, pro-tease, catalase, lysozyme, amylase, and lipase. Upon treatment, a decrease in antimicrobial activity was observed in the presence of protease, chymotrypsin, and trypsin, suggesting a proteinaceous nature of the antimicrobial compound. The zone of inhibition was measured at 16 mm for protease, 15 mm for chymotrypsin, and 18 mm for trypsin, while all other enzymes resulted in a consistent zone of inhibition of 19 mm. These findings underscore the susceptibility of the antimicrobial activity to enzymatic degradation by proteolytic enzymes, indicating its dependence on proteinaceous components for antimicrobial efficacy. Such insights are crucial for elucidating the biochemical nature of the antimicrobial compounds present in the CFS.
Morphological examination indicated that colonies of this active strain were circular, smooth, and elevated (Fig. 1B), with Gram staining confirming this Gram-positive nature (Fig. 1C). Scanning electron microscopy (SEM) analysis further corroborated the rod-shaped morphology of the strain (Fig. 1D).
Further characterization included catalase testing, which indicated that the isolated strain was catalase-negative, contrasting with the catalase-positive S. Typhi used as a control. Growth assessment under varied conditions encompassed different culture media, pH levels, salt concentrations (NaCl), and phenol concentrations. Among the media tested, MRS broth supported the most rapid growth of the isolated strain, with significantly lower growth observed in other media (Fig. 2A). Optimal growth occurred at pH 7.0 at 37°C (Fig. 2B), while exposure to increasing NaCl concentrations resulted in a progressive reduction in growth, with a notable decrease observed from 6.0% NaCl (Fig. 2C). The observed characteristics’ parallel findings reported in L. pentosus strains were isolated from naturally fermented Aloreña green table olives [80].
Evaluation of the strain’s tolerance to phenol revealed its ability to survive up to a 0.4% phenol concentration; however, it had diminished growth compared to control conditions, particularly at higher phenol concentrations (the growth decreased with increasing phenol concentration from 0.1 to 0.4% compared to the control) (Fig. 2D). These findings underscore the adaptability and resilience of the isolated strain to various environmental conditions.
The biochemical analysis indicated that the isolated bacteria could utilize all tested sugars, as outlined in Supplementary Table S1. It includes lactose, sucrose, fructose, glucose, sorbitol, mannitol, etc. Consistently, L. pentosus TEZU174 also exhibited a comparable sugar utilization profile [81]. Additionally, the results of various biochemical reactions, such as Voges-Proskauer’s test, citrate utilization, ONPG, and nitrate reduction, are detailed in Table 1.
Table 1
Biochemical test results of the L. pentosus strain.
Sl No. | Test | Principle | Results |
1. | Malonate | Malonate utilization | −ve |
2. | Voges Proskauer’s | Detects acetoin production | −ve |
3. | Citrate | Citrate utilisation | −ve |
4. | ONPG | Detects Beta galactosidase | +ve |
5. | Nitrate Reduction | Detects Nitrate reduction | −ve |
6. | Catalase | Detects Catalase activity | −ve |
7. | Arginine | Arginine utilisation | −ve |
8. | Esculin Hydrolysis | Detects Esculin Hydrolysis activity | +ve |
+ve: indicates positive results; −ve: negative results. |
Confirmation of its classification as a Lactiplantibacillus species was attained through 16S rRNA gene sequencing. Phylogenetic analysis unveiled a striking 99.6% similarity with strains, such as Lactiplantibacillus pentosus LMEM (MK240372.1), Lactiplantibacillus pentosus GCHI (MK245998.1), and several Lactiplantibacillus plantarum strains, including Lactiplantibacillus plantarum SCHI (MK246005.1), and Lactiplantibacillus plantarum MA8-6 (MG755354.1) (refer to Table 2 and Fig. 3).
Table 2
Microbial identification using 16S rRNA-based molecular method. Sequences of strains having significant alignments are given in the table.
Sl No. | Description | Identity | Accession No. |
1. | Lactiplantibacillus plantarum strain SCHI | 99.63% | MK246005.1 |
2. | Lactiplantibacillus pentosus strain GCHI | 99.63% | MK245998.1 |
3. | Lactiplantibacillus pentosus strain LMEM | 99.63% | MK240372.1 |
4. | Lactiplantibacillus plantarum strain MA8-6 | 99.63% | MG755354.1 |
5. | Lactiplantibacillus plantarum strain NWAFU1580 | 99.63% | MK045823.1 |
6. | Lactiplantibacillus plantarum strain TTF18 | 99.63% | MK028367.1 |
7. | Lactiplantibacillus plantarum strain TTF17 | 99.63% | MK028366.1 |
8. | Lactiplantibacillus plantarum strain TTF16 | 99.63% | MK028365.1 |
9. | Lactiplantibacillus plantarum strain TTF15 | 99.63% | MK028364.1 |
10. | Lactiplantibacillus plantarum strain TTF14 | 99.63% | MK028363.1 |
Further confirmation of its identity as Lactiplantibacillus pentosus (formerly known as Lactobacillus pentosus) strains was achieved through whole genome sequencing. Both datasets, including 16S rRNA gene accession (MN165450.1) and WGS GenBank assembly accession (GCA_009295675.1), were duly submitted to NCBI.
Genomic Characterization
The genomic analysis of Lactiplantibacillus pentosus reveals a composite structure consisting of 55 contigs (667,623 base pairs), with a total size of 3.7 Mb and a GC content of 46%. Further, 3342 coding sequences (CDSs) are identified within this genome, of which 3192 encode proteins. Comprehensive annotation further discloses the presence of 76 RNA genes, comprising 65 transfer RNA (tRNA), 7 ribosomal RNA (rRNA), and 4 non-coding RNA (ncRNA) genes.
Figure 4, generated through the CG View Server, provides a visual representation of the genome map, elucidating the arrangement of coding sequences on the strand, contigs, GC content, GC skew, and other pertinent genomic attributes. Additionally, supplementary tools enhance the analysis by identifying various genomic elements and properties. Specifically, Alien Hunter predicts putative Horizontal Gene Transfer (HGT) events, Phigaro detects prophage regions, Mobile OG-db identifies mobile genetic elements (MGEs), VirSorter discerns viral signals within microbial genomic data, CRISPR/Cas Finder detects CRISPR arrays along with their associated Cas proteins, and CARD facilitates the detection of antimicrobial resistance genes. Through this comprehensive approach, a thorough understanding of the genomic landscape of L. pentosus is achieved, enabling insights into its genetic makeup and functional potential
Bioinformatic Analysis
The probiotic characteristics of Lactiplantibacillus pentosus were meticulously investigated through blast analysis and manual examination of various proteins annotated by Prokka. This comprehensive investigation unveiled a spectrum of proteins implicated in stress response, adhesion, aggregation, resistance, and immunomodulation. Detailed information regarding these proteins and their NCBI accession numbers are provided in Table 3.
Table 3
Represents the details about the proteins present in the isolated L. pentosus strain genome that are involved in stress, adhesion, aggregation, and resistance with NCBI accession number.
Sl No. | Factors | Proteins | Accession No. |
1. | Stress | | |
| a. Temperature | cold-shock protein | MPQ17990.1, MPQ19628.1, MPQ20388.1 |
chaperonin GroEL | MPQ20734.1 |
co-chaperone GroES | MPQ20733.1 |
| | Hsp20 family protein | MPQ17905.1, MPQ18860.1, MPQ19037.1 |
| | Hsp33 family molecular chaperone HslO | MPQ20807.1 |
| b. pH | alkaline shock response membrane anchor protein AmaP | MPQ19576.1 |
| c. Other Stress | Universal stress protein | MPQ18033.1, MPQ18120.1, MPQ18171.1, MPQ18841.1, MPQ19344.1, MPQ19437.1, MPQ20001.1, MPQ20233.1, MPQ20274.1, MPQ20395.1 |
Peroxide stress protein YaaA | MPQ17946.1 |
GlsB/YeaQ/YmgE family stress response membrane protein | MPQ18166.1, MPQ19575.1 |
Asp23/Gls24 family envelope stress response protein | MPQ18230.1, MPQ18256.1, MPQ19578.1, MPQ19579.1 |
2. | Adhesion/Aggregation | | |
| Adhesion to mucus/epithelial cells/ECM proteins/plasma components/Aggregation | Mucus-binding protein | MPQ18214.1, MPQ20578.1 |
Chaperonin GroEL | MPQ20734.1 |
Elongation factor Tu | MPQ19535.1 |
Sortase | MPQ19087.1 |
Glyceraldehyde-3-phosphate dehydrogenase | MPQ19912.1 |
3. | Resistance | bleomycin resistance protein | MPQ17822.1 |
| | small multidrug resistance protein | MPQ17839.1 |
| | copper resistance protein CopZ | MPQ18979.1 |
Moreover, the genome-wide exploration for bacteriocin-encoding genes was conducted using BAGEL 4, revealing the presence of diverse bacteriocin types, including pediocin-like bacteriocin (class IIa), plantaricin E, F (class IIb), and a putative bacteriocin (Bovicin 255 variant). Pediocin showed similarity to bacteriocin from Pediococcus acidilactici. The Plantaricin E and F cluster, a two-peptide bacteriocin with immunity genes, ABC transporter genes, and accessory genes, exhibits similarity to bacteriocin from Lactiplantibacillus plantarum. Additionally, lactococcin is present within the same node. The putative bacteriocin (Bovicin 255 variant) shares similarities with the bacteriocin from Streptococcus mutans UA159. Notably, NCBI protein blast analysis affirmed the existence of garvicin Q family Class II bacteriocin and lactococcin (class IIc), demonstrating the capability of L. pentosus to produce a repertoire of Class II bacteriocins for defensive purposes. Figure 5A illustrates the outcomes obtained from BAGEL 4.
Similarly, other strains of L. pentosus exhibit distinct bacteriocin production profiles. For instance, L. pentosus ZFM94 synthesizes a bacteriocin termed Pentocin, while L. pentosus 124-2 produces two bacteriocins with molecular weights of 26.69 kDa and 17.15 kDa [82, 83].
Additionally, antiSMASH software was employed to conduct an in-depth analysis of secondary metabolite production, revealing the presence of two regions harboring secondary metabolites. Notably, region 6 encompasses a RiPP-like cluster (other unspecified ribosomally synthesized and post-translationally modified peptide product) containing a class II bacteriocin region with an ABC transporter and accessory protein, whereas region 32.1 harbors a T3PKS (Type III polyketide synthase) cluster with a hydroxymethylglutaryl-CoA synthase region. A cluster blast analysis of the bacteriocins cluster showed significant similarity to bacteriocin clusters of different L. plantarum strains, with identities ranging from 50 to 58% (Supplementary Figure S1 depicts the results of secondary metabolite search, and Figure S2 depicts the cluster blast results by antiSMASH). This describes the evolutionary relatedness in bacteriocin production. A similar cluster blast was used in L. pentosus strain ZFM94 [84].
The Plasmid Finder tool identified the presence of a plasmid in the genome with high identity to rep 28 from L. plantarum (NCBI accession number CP005948). This finding aligns with earlier reports indicating the presence of plasmids in certain L. pentosus strains [85].
The CRISPR Cas finder detected five sequences with CRISPR genes, indicating an adaptive immunity mechanism against foreign mobile genetic elements [86]. Nodes 1, 2, 7, 19, and 28 have CRISPR sequences. The genome has CAS-Type IIA and CAS-Type IE. Additionally, prophage regions were predicted by the PHASTER server, with nodes 13 and 15 containing complete and incomplete phage sequences, respectively (Fig. 5B). These findings are consistent with previous publications, suggesting that the genome of L. pentosus MP-10 included CRISPR Cas genes and prophage regions [87].
Furthermore, CARD analysis revealed the presence of the Van Y glycopeptide resistance gene cluster by strict hits with 29.63% identity (Fig. 5C). Loose hits (bit score below 500) showed the presence of 203 antibiotic resistance genes with different resistance mechanisms, including antibiotic efflux, antibiotic target protection, antibiotic inactivation, antibiotic target alteration, reduced permeability to antibiotics, and antibiotic target replacement. Resistance drug classes include glycopeptide antibiotics, peptide antibiotics, macrolide antibiotics, lincosamide antibiotics, cephalosporin, tetracycline antibiotics, aminoglycoside antibiotics, fluoroquinolone antibiotics, disinfecting agents, antiseptics, mupirocin-like antibiotics, rifamycin antibiotics, etc. These findings showed that the strain did not have rigorous antibiotic resistance.
These comprehensive analyses collectively enhance our understanding of the probiotic traits, bacteriocin production, genomic architecture, and antibiotic resistance profile of L. pentosus, underscoring its potential for therapeutic applications and biotechnological exploitation.
Probiotic Characterization
The L. pentosus strain underwent assessment for tolerance to bile and acid, revealing its capacity to withstand 0.15% bile salt concentration and acidic conditions with a pH of 3. The strain’s tolerance dynamics were investigated over time intervals of 0, 3, 6, 9, 12, and 24 h, with survivability percentages calculated from corresponding growth values, as depicted in Fig. 6A, B. Concurrently, cell viability was evaluated at these time points, and viable counts were recorded, as presented in Supplementary Table S2. Notably, the growth and cell viability levels of the Lactiplantibacillus pentosus strain exhibited an increasing trend over time intervals of 0, 3, 6, 9, 12, and 24 h. However, despite this increase, the percentage of survivability, when compared to the control, demonstrated a decreasing trend. This discrepancy suggests that while the bacterial population grows and maintains viability over time, its ability to survive in the presence of bile salt and acidic conditions decreases relative to the control group. This observation indicates that prolonged exposure to bile salt and acidic environments adversely affects the strain’s survivability, as evidenced by a reduction in its relative resilience compared to the control condition. This observation aligns with prior research conducted by Montoro et al., which explored the survivability of various L. pentosus strains under analogous conditions [80].
The culture of L. pentosus was screened for bile salt hydrolase (BSH) activity using a direct plate assay. BSH activity is recognized to facilitate bacterial colonization within the gastrointestinal tract [88]. Notably, the isolated L. pentosus strain produced a discernible white precipitate zone in bile agar media surrounding the colony, indicative of BSH production. Bifidobacterium adolescentis was the positive control used to validate this observation, while S. Typhi was employed as the negative control (Fig. 7A). Moreover, it is noteworthy that in earlier research, bile salt hydrolase (BSH) activity has been reported in several Lactiplantibacillus species originating from food and human sources. For instance, it has been observed that strains like L. pentosus CHIG have positive BSH activity [89].
Furthermore, the strain demonstrated 57.32% lysozyme tolerance with a CFU/mL count of 9.5 × 107 after 1 h of incubation. This finding underscores the strain’s capability to withstand lysozyme exposure.
Gastrointestinal tolerance of the isolated L. pentosus was assessed over intervals of 0, 3, 6, 9, 12, and 24 h. The results were similar to those observed for acid bile tolerance, with survivability percentages of the isolated strain in gastrointestinal conditions compared to control MRS documented in Fig. 6. Notably, an inverse relationship was observed between survivability percentage and time duration, wherein an increase in incubation time corresponded to a decrease in survivability percentage. The bacterial population is growing and maintaining viability over time; however, its ability to survive in the presence of gastrointestinal conditions decreases relative to the control group. Furthermore, Supplementary Table S2 provides a comprehensive overview of viable counts obtained at each time point, further elucidating the strain’s dynamics in gastrointestinal conditions. These findings underscore the strain’s ability to endure the challenges presented by the gastrointestinal environment. Such insights are pivotal for assessing the strain’s potential as a probiotic agent and its suitability for gastrointestinal health applications.
To evaluate its adhesion capability, the strain underwent testing for cell surface hydrophobicity using hydrocarbons, namely xylene, chloroform, and n-hexadecane. The observed hydrophobicity levels were 21.12 ± 0.94% in the presence of xylene, 13.53 ± 0.09% in the presence of n-hexadecane, and 26.72 ± 0.86% in the presence of chloroform. Comparable hydrophobicity profiles have been reported in other strains, such as Lactiplantibacillus pentosus isolated from fermented fish, Lactococcus lactis, and Lactobacillus fermentum [90].
Probiotic species’ radical scavenging capacity is an important characteristic, indicating their antioxidant nature. This was assessed using the DPPH radical scavenging assay, where the isolated L. pentosus demonstrated 60.70% DPPH activity, showcasing its antioxidative potential. Control ascorbic acid (10 µg/mL) gave 42.75% DPPH activity. Notably, studies by Unban et al. reported even higher DPPH scavenging activity in L. pentosus A14-6 and L. pentosus A26-8 [91]. A proteolytic assay was conducted using a skim milk agar plate, with trypsin (1.0 mg/mL) as the positive control, resulting in 19 mm of proteolysis. However, the supernatant and pellet of the isolated L. pentosus strain did not exhibit any zone of inhibition in the test, indicating the absence of proteolytic activities.
The antibiotic susceptibility profile of the L. pentosus isolate was assessed against a panel of 20 antibiotics, revealing resistance to cefoxitin (CX), amoxicillin/clavulanic acid (AMC), and amikacin (AK). However, sensitivity or intermediate resistance was observed towards all other antibiotics tested (refer to Table 4 and Fig. 7B, C), indicating the strain is susceptible to most antibiotics, thereby supporting its safety for probiotic applications [27]. This finding aligns with observations by Cazodo Munoz et al., who noted that out of 59 L. pentosus strains tested, a majority (95%) of strains were resistant to at least 3 antibiotics when evaluated against 15 antibiotics [92].
Table 4
Represents antibiotic susceptibility results of the isolated L. pentosus strain.
Sl No. | Antibiotics | Concentration (µg/Disc) | Zone of Inhibition (in mm) | Resistance/ Sensitive |
| Universal-1 OD308 | | | |
1. | Gentamicin (GEN) | 10 | 17 | I |
2. | Amikacin (AK) | 30 | 12 | R |
3. | Ciprofloxacin (CIP) | 5 | 15 | I |
4. | Cefoxitin (CX) | 30 | 0 | R |
5. | Amoxycillin/Clavulanic acid (AMC) | 20/10 | 11 | R |
6. | Tetracycline (TE) | 30 | 18 | I |
7. | Chloramphenicol (C) | 30 | 30 | S |
8. | Co-trimoxazole (COT) | 25 | 16 | I |
| Dodeca Universal-XII DE027 | | | |
9. | Ofloxacin (OF) | 5 | 16 | I |
10. | Cefadroxil (CFR) | 30 | 21 | S |
11. | Doxycycline HCl (DO) | 30 | 16 | I |
12. | Cloxacillin (COX) | 5 | 18 | I |
13. | Azithromycin (AZM) | 30 | 21 | S |
14. | Cefotaxime (CTX) | 10 | 20 | S |
15. | Ceftriaxone (CTR) | 30 | 20 | S |
16. | Ticarcillin (TI) | 75 | 17 | I |
17. | Piperacillin/ Tazobactam (PIT) | 100/10 | 25 | S |
18. | Ciprofloxacin (CIP) | 5 | 15 | I |
19. | Levofloxacin (LE) | 5 | 21 | S |
20. | Ceftazidime (CAZ) | 30 | 22 | S |
R = resistant (≤ 14 mm); S = sensitive (≥ 20 mm); I = intermediate (15–19 mm). |
A hemolytic assay was conducted for the safety assessment, wherein the cells and cell-free supernatant of the strain did not exhibit detectable hemolytic activity. However, the 10-fold concentrated cell-free supernatant showed media color diffusion (yellow to green color) with no clear zone of inhibition, indicative of potential partial hemolysis (α hemolysis) or absence of hemolysis. Triton x100 was used as the positive control, displaying a clear zone around the well (β hemolysis). In contrast, Nisin (1.0 mg/mL) was the negative control, demonstrating no hemolytic activity (Fig. 7D). Similar findings were reported for L. pentosus 22C, isolated from traditional yogurt, which also lacked hemolytic activity [93]. The absence of clear hemolytic (β) activities confirms that the strain is safe to use.
Furthermore, the strain exhibited 23% auto-aggregation (Fig. 8A) and 51% co-aggregation with S. Typhi (Fig. 8B). Previous reports have indicated that L. pentosus exhibits notable co-aggregation abilities, including solid co-aggregation with Streptococcus mutans [94]. Many L. pentosus strains isolated from naturally fermented Aloreña table olives exhibited similar auto-aggregation and co-aggregation. Among them, 19% of L. pentosus strains had a high capability to auto-aggregate (50–77.92%), while 42% had a medium auto-aggregation capacity (35–50%). They also showed different ranges of co-aggregation with pathogens, including E. coli, Salmonella, Listeria innocua, and Staphylococcus aureus [80].
Moreover, the antibacterial activity of the cell-free supernatant (CFS) was evaluated using an agar-well diffusion assay, revealing antibacterial activity against various pathogens, including both Gram-positive and Gram-negative strains. Notably, in some instances, the antibacterial activity of the CFS was comparable to streptomycin (antibiotic) and superior to nisin (commercially available bacteriocin). Further details are provided in Table 5. These observations are consistent with findings reported for L. pentosus strains provided by CICC (China Centre of Industrial Culture Collection) [95] and L. pentosus ZFM94, isolated from infant faces [82]. These evaluations collectively offer insights into various functional characteristics of L. pentosus, contributing to a comprehensive understanding of its probiotic attributes.
Table 5
Represents the antibacterial activity of CFS of the isolated L. pentosus strain against different food pathogens.
Sl No. | Bacteria | ZOI Cell-Free Supernatant in mm | ZOI Nisin in mm | ZOI Streptomycin in mm |
1. | Listeria monocytogenes | 19 | 10 | 19 |
2. | Vibrio harveyi | 16 | 14 | 13 |
3. | Streptococcus mutans | 16 | 13 | 13 |
4. | Staphylococcus aureus | 16 | 10 (not clear) | 16 |
5. | Streptococcus thermophilus | 18 | 0 | 14 |
6. | Corynebacterium callunae | 20 | 11 | 20 |
7. | Enterococcus gallinarum | 18 | 14 | 19 |
8. | Vibrio cholerae | 16 | 0 | 14 |
9. | Bacillus cereus | 18 | 13 | 25 |
10. | Pseudomonas putida | 16 | 0 | 19 |
11. | Escherichia coli | 16 | 0 | 20 |
12. | Pseudomonas aeruginosa | 16 | 10 | 16 |
13. | Clostridium perfringens | 21 | 0 | 16 |
14. | Salmonella enterica subsp. enterica ser. Typhi | 18 | 0 | 16 |
15. | Salmonella enterica subsp. enterica ser. Paratyphi | 17 | 0 | 17 |
ZOI is the zone of inhibition, and CFS is the cell-free supernatant of the isolated L. pentosus strain. |