Basic probiotic testTolerance to pepsin

Tolerance of MKMB01 and MKMB02 to gastrointestinal conditions such as pepsin, pancreatin, and ox gall (bile) was determined by measuring viable cell counts, represented in log10 CFU/mL in Supplementary Fig. 1a, b, and c. Compared to the control (PBS, pH 7.4), the viability of MKMB01 and MKMB02 decreased significantly after 3 hours of treatment with pepsin (PBS, pH 2) but retained their viability to 90.10% ± 1.07 (p ≤ 0.001) and 82.95% ± 0.69 (p ≤ 0.001) respectively (Supplementary Fig. 1a). After exposure to pancreatin (pH 8, 1 mg/mL pancreatin) and 2% (w/v) bile salt, oxgall (pH 7.4) for 4 hours, both of the bacterial strains retained their viability with no significant differences as compared to the control (pH 7.4) (Supplementary Fig. 1b and c). These results indicate that the MKMB01 and MKMB02 strains can thrive in harsh gastrointestinal conditions with high viability percentages (Supplementary Table 1). Representative images of the colony forming units (CFU) on agar plates are presented in Supplementary Fig. 2A, B, C, D, and E.

Antibiotic susceptibility and antimicrobial activity

The bacterial strains MKMB01 and MKMB02 were tested for antibiotic susceptibility against common antibiotics using Hexa G plus antibiotic discs (Himedia, India) (Supplementary Fig. 3A and B) containing ampicillin (AMP = 10 µg), chloramphenicol (C = 25 µg), penicillin (P = 1 unit), streptomycin (S = 10 µg), sulphatriad (S3 = 300 µg), and tetracyclin (TE = 25 µg). As presented in Supplementary Table 2, MKMB01 and MKMB02 are resistant to streptomycin, but they are moderately susceptible to other antibiotics. Images of the culture plates are shown in Supplementary Fig. 3A and B.

The antimicrobial properties of both strains were also tested using culture supernatants against pathogens such as E. coli K12, K. pneumoniae, L. monocytogenes, S. typhimurium, S. aureus, and E. faecalis. MRS broth was used as the control, and no antimicrobial activity was observed. The interpretation of the antibiotic and antimicrobial susceptibility test results follow the guidelines recommended by the Clinical & Laboratory Standards Institute (CLSI); i.e., zone of inhibition (ZOI) ≤ 14 mm as resistant (R), 15–19 mm as intermediate, and ≥ 20 mm as susceptible [48, 49]. Antibiotic susceptibility testing was carried out on Mueller–Hinton agar. Based on the results, MKMB01 and MKMB02 possess low antimicrobial activity, with the highest activity against K. pneumoniae (18.66 ± 0.8 and 18 ± 0) and S. aureus (18.66 ± 0.8, and 18.33 ± 0.6). The lowest activity was observed in E. faecalis (15 ± 0 and 15.66 ± 0.6) (Supplementary Fig. 3C-I and Supplementary Table 3).

Additional probiotic propertiesSurface hydrophobicity and auto-aggregation ability

Surface hydrophobicity and auto-aggregation were evaluated with regards to the adhesion ability in the gut (Supplementary Fig. 1d and e). Our study demonstrated that MKMB01 (52.5%, p ≤ 0.001) has a higher surface hydrophobicity than MKMB02 (32.3%%V, p ≤ 0.001). Both MKMB01 and MKMB02 strains possess medium auto-aggregation ability (68.7%; p ≤ 0.001 and 42.1%; p ≤ 0.001, respectively) (Supplementary Fig. 1e) (Reference range: High is ≥ 70% AA, Medium is 20–70% AA, and Low is ≤ 20% AA; [25]).

Adhesion to HT-29 cells

Adhesion to the epithelia of the gut lining is essential for exerting the beneficial effects of probiotics. The adhesion ability of the MKMB01 and MKMB02 strains was evaluated by incubating with a monolayer mucin-producing colonic HT-29 cell line at 1 × 109 CFU/mL for 4 hours. Both stains demonstrated high adhesion capacity to the monolayer; however, MKMB01 had a higher adhesion ability than MKMB02 (54.5% vs. 23.8%; p ≤ 0.001 and p ≤ 0.001, respectively) (Supplementary Fig. 1F).

MKMB01 and MKMB02 adhesion to Ht-29 cells (Supplementary Fig. 4A, a, b, and c; and B, a, b, and c) was visualized using field emission scanning electron microscopy (FESEM). We found that both L. plantarum strains (MKMB01 and MKMB02) adhered to HT-29 cells.

The formation of biofilms was also monitored using FESEM. We observed that MKMB01 and MKMB02 strains formed biofilms on glass coverslips in MRS broth when cultured 48 hours at 37 °C (Supplementary Fig. 4 C, a, b, and c; and D a, b, and c. The scale bars in a, b, and c denote 20 μm/5KX, 6 μm/10KX, and 2 μm/30KX, respectively).

Antioxidant activity

Intracellular antioxidant activity was evaluated using cellular extracts of the two probiotic bacteria MKMB01 and MKMB02 (Supplementary Fig. 5). MKMB01 possessed DPPH radical scavenging potential of 29.42%± 0.73 (p ≤ 0.001), whereas MKMB02 of about 26.84 ± 0.47 (p ≤ 0.001).

Effect of MKMB01 and MKMB02 on maintaining gut integrity

The potential effect of MKMB01 and MKMB02 on the expression of tight junction proteins (TJPs) was assessed using caco-2 cells and confocal microscopy. The abundance and localization of TJPs like claudin 1, ZO-1, and occludin were visualized (Fig. 1A, a-d; B, a-d; and C, a-d) and presented graphically in Fig. 1Ae, Be, and Ce. The control caco-2 cells (without treatment) had uniform borders around each cell. In contrast, Salmonella-treated cells (NEG) lost cellular integrity and well-defined uniform borders, and the expression of claudin-1, ZO-1, and occludin were estimated to be reduced to 34.39% ± 3.49 (p ≤ 0.001), 38.33% ± 2.87 (p ≤ 0.001), and 54.16% ± 3.3 (p ≤ 0.001), respectively.

Prior treatment with MKMB01 for 4 hours reduced the severity of Salmonella invasion by maintaining the expression of claudin-1, ZO-1, and occludin to 85.37% ± 4.39 (p ≤ 0.001), 77.85% ± 5.58 (p ≤ 0.001), and 85.845% ± 6.51 (p ≤ 0.002), respectively. Similarly, MKMB02 also maintained the expression levels of claudin-1, ZO-1, and occludin to 83.51% ± 1.73 (p ≤ 0.001), 64.37% ± 2.86 (p ≤ 0.001), and 70.13% ± 2.38 (p ≤ 0.03) respectively. Thus, treatment with MKMB01 and MKMB02 for 4 hours before Salmonella treatment maintained the expression of TJPs in caco-2 cells, to reduce disease severity.

Our study also investigated the effect of MKMB01 and MKMB02 on MUC-2 gene expression in HT-29 cells. Incubation with MKMB01 and MKMB02 before Salmonella treatment in HT-29 cells increased the MUC-2 gene expression by ≈ 2 folds (p ≤ 0.001) and 1 fold (p ≤ 0.003), compared to cells treated with Salmonella alone, which showed ≈ 1 fold decrease in MUC-2 gene expression (p ≤ 0.005).

We also monitored the integrity of the intestinal barrier by evaluating the TEER of MKMB01 and MKMB02 strains in Salmonella-challenged caco-2 cells. TEER measurements are non-invasive in monitoring the integrity of the epithelial or endothelial cell layer barrier. Caco-2 cells express a variety of TIJs and connect with neighboring cells, allowing ions to flow through para-cellular and trans-cellular pathways. Therefore, TEER reflects the barrier function and can indicate gut integrity. With Salmonella, TEER was reduced to 286.50 ± 17.66 Ω/cm2 in 6 hours from 801.00 ± 11.23 Ω/cm2 at 0 hour. Pre-treatment with MKMB01 and MKMB02 strains before Salmonella administration attenuated the severity of Salmonella-induced barrier disruption and increased TEER by ≈ 0.7 fold (p ≤ 0.001) and 0.8 fold (p ≤ 0.001), respectively, after 6 hours, compared to Salmonella treatment alone (Fig. 1E).

Fig. 1

Effect of L. plantarum stains MKMB01 and MKMB02 on gut integrity. Representative confocal images of tight junction protein (TJP) expression with Salmonella-infected caco-2 cells are shown as (A) claudin-1, (B) ZO-1, and (C) occludin; a, b, c, and d denote control, negative control, MKMB01, and MKMB02, respectively for each group. Graphical representation of TJP expression is represented by (e). (D) RT-PCR result of MUC-2 gene expression and (E) TEER assay. Changes in TEER with time are compared with negative control. Values are shown as mean (± SEM) from three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups whereas #between Salmonella treated and control groups). Scale bar: 25 μm

Effect of MKMB01 and MKMB02 on inflammatory and anti-inflammatory markersProbiotic treatment reduced the release of nitric oxide (NO) by Salmonella-induced RAW 264.7 cells

The release of NO was determined using THP-1 and RAW 264.7 cells. Treatment with Salmonella did not significantly induce NO in THP-1 (data not presented). Therefore, we further tested with RAW 264.7 cells. Unlike THP-1 cells, with the treatment of Salmonella RAW 264.7 cells released NO into the culture medium after Salmonella treatment reaching concentration of 26.53 ± 0.55 µM in 8 hours, from a concentration of 4.18 ± 0.27 µM at 0 hour (p ≤ 0.001) (Fig. 2AP). Pre-treatment with MKMB01 and MKMB02 for 4 hours before Salmonella infection reduced NO concentration by 15.29 ± 0.1 µM (p ≤ 0.001) and 12.5 ± 0.05 µM (p ≤ 0.001), respectively, after 8 hours (Fig. 2A).

Fig. 2

Immunoregulatory effect of L. plantarum MKMB01 and MKMB02. Graphical representation of the genes (A) NO, (B) IL-1β, (C) MCP-1, (D) SIGIRR, (E) TOLLIP, (F) IRAK-M, (G) A20, (H) IL-10, (I) TGF-β, (J) IL-6,(K) TNF-α, (L)), and (M)). TNF-α release by THP-1 and HT-29 cells in pg/mlmL; (N) and (O) IL-6 release by THP-1 and HT-29 cells in pg/mlmL; (P) ADAM10, (Q) ADAM17, (R) iRhom-1, (S) iRhom-2, and (T) ADAM17 expression in THP-1 cells (a, b, c, and d denote control, negative control, MKMB01, and MKMB02, respectively for each group), ); and (U) graphical representation of ADAM17 expression. Changes in gene/protein expression were compared with untreated control and Salmonella treatment. Values are shown as mean (± SEM) from three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups, whereas #between Salmonella treated and control groups). Scale bar: 25 μm

MKMB01 and MKMB02 reduced the expression of IL-1β and inflammatory chemokine MCP-1 in Salmonella-induced caco-2 cells

IL-1β is an essential inflammatory cytokine mediator of inflammatory responses involved in various gut-associated disorders. With Salmonella treatment, IL-1β mRNA expression increased by ≈ 2.3 folds (P ≤ 0.001) compared to the untreated control. Following pre-treatment of MKMB01 and MKMB02 strains for 4 hours prior to Salmonella treatment, the IL-1β mRNA level was reduced ≈ 2.2 folds (P ≤ 0.001) and 1.7 folds (P ≤ 0.001), respectively ((Fig. 2B).

On the other hand, pre-treatment with MKMB01 and MKMB02 also reduced the expression of MCP-1 by ≈ 0.75 fold (p ≤ 0.001) and by ≈ 0.42 fold (p ≤ 0.002), respectively, which was elevated by ≈ 0.96 fold (p ≤ 0.001) when stimulated with Salmonella, thus minimizing the Salmonella-induced inflammatory responses in caco-2 cells.

Lactiplantibacillus strains MKMB01 and MKMB02 modulate the expression of TLR-negative regulators in caco-2 cells

In the literature, Salmonella infection is reported to trigger the expression of TLR-4, playing an essential role in activating host innate and adaptive immunity. Therefore, we investigated the potential of MKMB01 and MKMB02 to modulate the expression of TLR-negative regulators. Surprisingly, treatment with Salmonella alone did not alter the expression of TLR-negative regulators, such as toll-interacting protein (Tollip) and TNF-α-induced protein 3 (TNFAIP3) (also known as A20) (Fig. 2Eand G). Nevertheless, MKMB01 and MKMB02 pre-treatment increased the expression of Tollip significantly (Fig. 2E) by ≈ 0.9 fold (p ≤ 0.004) and ≈ 1.02 fold (p ≤ 0.002), respectively. Similarly, A20, which is a critical negative regulator of NF-κB, increased significantly (Fig. 2G) by ≈ 0.69 fold (p ≤ 0.045) and ≈ 0.53 fold (p ≤ 0.043)), respectively.

On the other hand, single Ig IL-1-related receptor (SIGIRR), also known as toll/interleukin-1 receptor 8 (TIR8) was reduced by ≈ 0.4 fold after Salmonella treatment (Fig. 2D). This effect was altered with MKMB01 and MKMB02 pre-treatment (Fig. 2D) which increases the expression of SIGIRR by ≈ 1.71 folds (p ≤ 0.001) and 1.5 folds (p ≤ 0.001), respectively. In contrast, the expression of interleukin-1 receptor-associated kinase (IRAK)-M showed no changes with either treatment (Fig. 2F).

MKMB01 and MKMB02 regulate the expression of anti-inflammatory cytokines TGF-β and IL-10

Releasing high levels of TGF-β and IL-10 suppresses pathological immune responses and promotes tolerance in the gut. Therefore, we tested MKMB01 and MKMB02 for their potential to regulate TGF-β and IL-10 expression in Salmonella-stimulated caco-2 cells. Compared to untreated control and Salmonella-treated cells, mRNA expression of IL-10 was reduced by ≈ 0.4 fold (p ≤ 0.043), but no significant change was seen in the expression of TGF-β (Fig. 2H and I). Nevertheless, pre-treatment of MKMB01 and MKMB02 for 4 hours before the Salmonella infection increased the expression of IL-10 (Fig. 2H) by ≈ 1.8 folds (p ≤ 0.001) and 1.5 folds (p ≤ 0.001), respectively, compared to the untreated control and cells treated with Salmonella alone. Similarly, pre-treatment of MKMB01 and MKMB02 increased the expression of TGF-β to ≈ 1 fold (p ≤ 0.001) and 1.5 folds (p ≤ 0.001), compared to Salmonella treated cells (Fig. 2I).

Effect of MKMB01 and MKMB02 on the expression of proinflammatory cytokines TNF-α and IL-6

TNF-α and IL-6 play crucial roles in immune responses and antigen presentation. To analyze the potential of MKMB01 and MKMB02 in reducing the expression of proinflammatory cytokines at transcriptional and protein levels, PMA-differentiated THP-1 cells were co-cultured with caco-2 cells and pre-treated with MKMB01 and MKMB02, which were then infected with Salmonella for 6 hours. Pre-treatment of MKMB01 and MKMB02 decreased the mRNA expression of TNF-α (Fig. 2J) by ≈ 1.45 folds (p ≤ 0.001) and 1.04 folds (p ≤ 0.001), respectively, compared to Salmonella treatment alone, which increased by 0.89 fold (p ≤ 0.001) compared to the control. Similarly, with MKMB01 and MKMB02 pre-treatment, the mRNA expression of IL-6 (Fig. 2K) decreased by ≈ 0.42 fold (p ≤ 0.007) and 0.33 fold (p ≤ 0.014), respectively, compared to cells treated with Salmonella alone, which increased the expression by ≈ 0.2 fold (p ≤ 0.04), compared to control.

We also assessed the release of soluble TNF-α and IL-6 into the culture media in PMA-differentiated THP-1 cells co-cultured with caco-2 cells (to depict intestinal conditions). We found that treatment with Salmonella increased the release of soluble TNF-α (Fig. 2L) to a concentration of 900.64 ± 31.26 pg/mL (p ≤ 0.001), compared to the control (19.66 ± 2.93 pg/mL). MKMB01 and MKMB02 pre-treatment; however, reduced the release of soluble TNF-α to 304.60 ± 39.02 pg/mL (p ≤ 0.001) and 483.74 ± 35.74 pg/mL (p ≤ 0.001), respectively. Interestingly, the release of IL-6 (Fig. 2N) into the culture media reached only up to 20.99 ± 1.77 pg/mL (p ≤ 0.001) with Salmonella treatment, compared to control (3.01 ± 0.61 pg/mL). With MKMB01 and MKMB02 treatments, the concentration of IL-6 was reduced to 3.03 ± 0.5 pg/mL (p ≤ 0.001) and 2.85 ± 0.73 pg/mL (p ≤ 0.001), respectively, compared to THP-1 cells with Salmonella-treatment.

We also tested for TNF-α and IL-6 release using caco-2 and HT-29 cells, but found that caco-2 cells do not produce either of the cytokines. HT-29 released lower concentrations of TNF-α and IL-6 (Figs. M and O), compared to THP-1 cells. With Salmonella treatment, the concentration of TNF-α in the culture media increased to 394.34 ± 17.00 pg/mL (p ≤ 0.001), compared to the control (1.45 ± 0.86 pg/mL). MKMB01 and MKMB02 treatments reduced the concentrations to 190.83 ± 1.38 pg/mL (p ≤ 0.001) and 194.28 ± 2.76 pg/mL (p ≤ 0.001), respectively. With Salmonella treatment, the concentration of IL-6 increased to 8.63 ± 1.07 pg/mL (p ≤ 0.002), compared to the control (0.19 ± 0.00 pg/mL. Following MKMB01 and MKMB02 treatments; however, the concentrations were reduced to 1.10 ± 0.32 pg/mL (p ≤ 0.002) and 2.10 ± 0.45 pg/mL (p ≤ 0.002).

MKMB01 and MKMB02 affect the release of proinflammatory cytokines by regulating the expression of ADAM17 protein

We studied the effects of MKMB01 and MKMB02 in regulating the expression of inflammatory regulators and adaptors (ADAM10/ADAM17) using RT-PCR followed by immunocytochemistry. Even though ADAM10 and ADAM17 formed with similar sequences and structures, only ADAM17 was expressed (Fig. 2P and Q). Salmonella treatment increased the mRNA expression of ADAM17 by ≈ 0.61 fold (p ≤ 0.001) compared to control which was reduced by MKMB01 and MKMB02 treatment by ≈ 0.80 fold (p ≤ 0.001) and 0.60 fold (p ≤ 0.001), respectively. Therefore, we proceeded to examine the expression of the ADAM17 protein. Similar to the mRNA results, the expression of the ADAM17 protein increased with Salmonella infection by ≈ 2.8 folds (p ≤ 0.001) compared to the control (Fig. 2T (a, b, c, and d) and U). With MKMB01 and MKMB02 pre-treatment, the expression of ADAM17 was reduced by ≈ 1.87 folds (p ≤ 0.001) and 1.53 folds (p ≤ 0.001), compared to Salmonella treatment alone. Thus, MKMB01 appeared to have a greater potential to reduce the release of soluble TNF-α and its adaptor protein ADAM17.

ADAM17 is an enzyme that requires proteolytic cleavage for activation and trafficking by rhomboid proteins (iRhoms) (i.e., iRhom1 and 2). The qRT-PCR results indicated that pre-treatment of MKMB01 and MKMB02 significantly decreased the expression of iRhom2 (≈ 1.26 folds, p ≤ 0.001 and 0.83 fold, p ≤ 0.001, respectively) compared to Salmonella treatment, which increased the expression by ≈ 1.52 folds (p ≤ 0.001), compared to the control (Fig. 2S). Nevertheless, we found no significant change in the expression of iRhom1 in any treatments (Fig. 2R).

MKMB01 and MKMB02 reduced Salmonella-induced apoptosis in caco-2 cells

During final phases of apoptosis, cells lose their membrane asymmetry, which leads to the translocation of membrane phospholipids such as phosphatidylserine to the outer layer of the plasma membrane. This relocation can be detected by its strong affinity to Annexin V and DNA-binding dyes such as PI. In untreated caco-2 cells, 87.9% of the cells were viable/live cells, 6.48% were pre-apoptotic, 4.97% were apoptotic, and a negligible amount of ≈ 0.65% were necrotic cells (Fig. 3A and E). After Salmonella treatment (Fig. 3B and E), the viability of caco-2 cells was reduced to 40.86% (p ≤ 0.001), with apoptotic cells increasing to 49.41% (p ≤ 0.001), and necrotic cells increasing to 6.44% (p ≤ 0.001). No significant changes were observed in pre-apoptotic cells.

The decreased viability of the caco-2 cells following Salmonella treatment was reversed with MKMB01 and MKMB02 pre-treatment (Fig. 3C, D, and E) to 79.51% (p ≤ 0.001) and 80.88% (p ≤ 0.001), respectively. Similarly, apoptotic cells were reduced to 9.86% (p ≤ 0.001) and 9.97% (p ≤ 0.001), respectively, with no significant changes in pre-apoptotic and necrotic caco-2 cells, compared to Salmonella treatment.

Fig. 3

Effect of L. plantarum MKMB01 and MKMB02 on apoptosis, ROS generation, and mitochondrial function. (A-E) denote apoptosis [Upper left quadrants = necrotic cells (P1-1), upper right quadrants = apoptotic cells (P1-2), lower left quadrants = viable cells (P1-3), and lower right quadrants = pre-apoptotic cells (P1-4)]; (F-J) denotes ROS generation (P2 = ROS negative and P3 = ROS positive); (K) Mitochondrial respiration; (L) Non-mitochondrial respiration; (M) Basal respiration; (N) Maximal respiration; (O) Spare respiratory capacity (SRC) and (P) ATP Production. Values are expressed as means (± SEM) for three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups, whereas #between Salmonella treated and control groups)

MKMB01 and MKMB02 reduced Salmonella-induced ROS generation in caco-2 cells

We analyzed the potential of MKMB01 and MKMB02 in reducing the ROS generated in caco-2 cells from Salmonella infection. As a result of Salmonella infection, ROS increased to 32.7% (p ≤ 0.001) as compared to control (3.53%). With MKMB01 and MKMB02 pre-treatment; however, cellular ROS generation was reduced to 7.98% (p ≤ 0.001) and 15.33% (p ≤ 0.001), respectively (Fig. 3F-J).

MKMB01 and MKMB02 reduced Salmonella-induced mitochondrial dysfunction

In the experimental results shown in Fig. 3F-J, Salmonella-induced oxidative stress and ROS generation disrupted mitochondrial and cellular functions, leading to cell death. Nevertheless, the pre-treatment of MKMB01 and MKMB02 significantly reduced Salmonella-induced ROS generation in the treated cell lines. In this regard, we evaluated the cellular bioenergetics and mitochondrial functioning of caco-2 cells with MKMB01 and MKMB02 in reducing oxidative stress aginst Salmonella-treated cells (Fig. 3).

The oxygen consumption rate in real-time was measured using an XFe24 extracellular flux analyzer. Functional parameters like basal respiration, non-mitochondrial respiration, and ATP production of the Salmonella-treated cells were significantly reduced from 356.19 ± 1.94 (p ≤ 0.001), 72.30 ± 1.94 (p ≤ 0.001), and 289.17 ± 3.20 (p ≤ 0.001) to 808.48 ± 10.87, 130.94 ± 1.97 and 664.85 ± 13.94, respectively, compared to control. Similarly, maximal respiration and spare respiratory capacity (SRC) were also reduced following Salmonella infection (Supplementary Table 5). Treatment with MKMB01 and MKMB02 significantly restored all of the above parameters i.e., basal respiration (619.68 ± 9.52 and 520.86 ± 11.92 (p ≤ 0.001 and p ≤ 0.001)); non-mitochondrial respiration (100.53 ± 3.76 and 91.14 ± 91.14 (p ≤ 0.002 and p ≤ 0.007)); ATP production (510.7 ± 8.7, and 418.6 ± 8.6 (p ≤ 0.001 and p ≤ 0.001)); maximal respiration (1190.6 ± 6.1, and 827.0 ± 82.1 (p ≤ 0.001 and p ≤ 0.001)); and SRC (567.56 ± 3.50, and 436.10 ± 6.19 (p ≤ 0.001 and p ≤ 0.001)).

Whole genome study (WGS) of MKMB01 and MKMB02Genomic features

The complete genome of MKMB01 was sequenced using the Illumina Miseq platform, and initial data was obtained. After removing the adapter sequences, 99.14% reads were retained. The genome size of MKMB01 was found to be 3.06 Mb with 44.56% GC content. The annotation pipeline detected the presence of 2875 coding sequences out of 2936 genes in the MKMB01 genome. In addition, genes related to 4 ribosomal RNA and 56 transfer RNA were identified (Fig. 4). The genome size of MKMB02 was 3.37 Mb, and the average GC content was 44.01%. The analysis revealed 3246 genes, 3183 coding sequences, 4 ribosomal RNA, and 58 transfer RNA in the MKMB02 genome (Supplementary Table 3). All WGS data was deposited in the NCBI database with accession no. JBDLNZ000000000 for MKMB01, and JBDPJJ000000000 for MKMB02.

Fig. 4

The whole genome sequences of the MKMB01 and MKMB02 probiotic strains. The genomic sequences of MKMB01 (A) and MKMB02 (B) reveal their functional capabilities (C & D). Putative bacteriocin ORFs (antimicrobial peptides) were detected (E & F)

Assessment of probiotic characteristics

The two genomes were screened for genes related to probiotic attributes, including acid tolerance, adhesion, stress response, and immunomodulation (Table 1). Details of the WGS analysis are presented in Supplementary 3.

Table 1 Genes present in the MKMB01 and MKMB02 genomes