Abstract Pla-som is a product from traditional food preservation method that has grown to become a popular product in Thailand's Phayao Province. In the current study, lactic acid bacteria (LAB) were isolated and assessed for their probiotic properties. A total of 20 isolates were selected and based on morphological and some biochemical testing. Among these, 2 strains, PY13 and PY 41, were able to survive at pH 2.0, 7% NaCl and 0.3% bile salt conditions. These strains were further evaluated for antibiotic susceptibility, antagonistic activity, hemolysis activity, aggregation activities and β-galactosidase production. The results showed that both strains possessed an aggregation activity, β-galactosidase, non-hemolysis activity, and inhibition to the growth of Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The antibiotic susceptibility test showed susceptibility to most tested antibiotics except for vancomycin and ciprofloxacin. The selected LAB strains were identified by 16S rRNA sequencing as Lactiplantibacillus pentosus.  The study demonstrated the probiotic potential of isolated LAB strains, suggesting that they might be used as food products and dietary supplements.

Keywords: Lactic acid bacteria, Pla-som, Probiotic properties, Thai fermented fish

Funding: This research project was supported by the Thailand Science Research and Innovation Fund and University of Phayao (Fundamental Fund 2023; FF66-RIM081).

Citation:  Supadej, K., Khamfan, N., Doosorn, B., Jongruamklang, P., Kummasook, A., and Suradej, B. 2026. Identification and probiotic characterization of Lactiplantibacillus pentosus isolated from pla-som, a traditional Thai fermented fish of Phayao Province, Thailand. Natural and Life Sciences Communications. 25(3): e2026068.

Graphical Abstract:

INTRODUCTION

Pla-som is a type of traditional food preservation that has been developed into a well-known product from Phayao Province, Thailand. Pla-som recipes vary depending on the region and local consumer preferences. This product is made from whole fish or fish fillets and fermented with cooked or sticky rice, garlic, and salt. The process results in a sour taste initiated by natural microorganisms, mainly lactic acid bacteria (LAB) (Kopermsub and Yunchalard, 2010). Fermented foods are a beneficial source of good microorganisms. Traditional fermentation processes and varying environmental conditions enhance the diversity and quality of microorganisms.

LAB is a commonly found microorganism in fermented products and is particularly important in food preservation. LAB includes a large number of genera, which are Gram-positive, non-spore forming, and catalase negative rods and cocci bacteria (Zapaśnik et al., 2022). They are able to cause fermentation of food stuffs through the production of lactic acid. Additionally, the fermentation process lowers the natural pH, making it difficult for pathogenic microorganisms to develop. LAB also produces metabolites with antimicrobial effects such as organic acid, hydrogen peroxide, diacetyl, inhibitory enzymes, and bacteriocins (Elayaraja et al., 2014). Some LAB and their produced metabolites are “Generally Considered As Safe” (GRAS) for the food industry (Simons et al., 2020). Recently, probiotics have been promoted as healthy supplements that improve the intestinal microbial balance of the human host. Probiotics are defined as living microorganisms that provide health benefits to humans when administrated in adequate numbers according to Guideline for the Document Preparation for Safety Evaluation of Probiotics in Food (National Center for Genetic Engineering and Biotechnology [BIOTEC], National Science and Technology Development Agency [NSTDA]). LAB genera are generally recognised as probiotic strains and considered to be good candidates for industrial and medical prospects (Pavunc et al., 2023; Srinivash et al., 2023). Probiotic bacteria demonstrate a desirable impact on reducing blood cholesterol levels, stimulation of the immune system, and reducing the risk of carcinogenesis (Dunne et al., 2001). Furthermore, probiotics play a significant role in the gastrointestinal system by inhibiting pathogenic microorganism adhesion, producing antibacterial substances, vitamin synthesis, and producing metabolites that regulate homeostasis (Ng et al., 2008; Zapaśnik et al., 2022).

In Thailand, probiotic products were widely available as commercial formulations for human consumption and market growth is expected. In the future, probiotic formulations, dietary supplements, and functional foods are predicted to be in demand for the treatment of a variety of illnesses (Floch, 2014; Anjum and Ouwehand, 2023). Foods that have undergone fermentation contain healthy microbes. Environmental factors and the traditional fermentation procedure used in the area improve the microbial diversity and quality of fermented foods. According to Sanpa et al. (2019), LAB strains are rich and diverse in Pla-som. However, a significant research gap exists regarding the probiotic potential of LAB strains indigenous to Pla-som from Phayao, Northern Thailand. This region’s specific environmental factors and traditional techniques may harbor unique strains with superior functional properties. Therefore, this study aims to isolate and characterize LAB from Phayao’s Pla-som to identify high-potency candidates. Characterizing these strains is essential in developing standardized starting cultures that can connect traditional food knowledge with the commercial manufacturing of high-value functional foods or nutraceuticals.

MATERIALS AND METHODS

Isolation of lactic acid bacteria

Three different kind of Traditional fermented fish products or Pla-som, pure fish fillet, fish fillet with skin, and whole fish were purchased from a producer in Phayao province, Thailand. The fermentation time of all products was between 3-5 days (Sriphannam and Kummasook, 2020). To identify the LAB, 10 g of samples were homogenized, with 90 mL of sterilized 0.1% (w/v) peptone solution and serially diluted. Then, the diluted sample was plated onto de Man Rogosa Sharp (MRS) agar (Hi-media, India) containing 0.4% (w/v) bromocresol purple. Plates were incubated anaerobically at 37°C for 48 hours using a candle jar (Kopermsub and Yunchalard 2010; Sanpa et al., 2019). Colonies changed to yellow were selected and collected for characterization of their morphology by Gram stain and some biochemical tests including catalase and Oxidation–Fermentation (OF) test. Gram-positive and catalase negative strains were maintained on MRS broth with 20% glycerol at -80°C.

Probiotic properties of LAB

Acid tolerance

As previously described by Sriphannam and Kummasook (2020), the LAB cells were grown in MRS broth at 37°C for 18 hours and then sub-cultured in 10 mL of PBS, which was adjusted to pH 2.0. The initial bacteria concentration was 1.0 × 10⁸ CFU/mL, and the LAB was incubated for 3 hours at 37°C. The culture without pH adjustment served as a control. The LAB variable growth was observed with MRS supplemented with 0.02% bromocresol purple after 48 hours of incubation.

NaCl tolerance test

Overnight LAB cultures were inoculated into MRS broth adjusted with different concentrations of NaCl (1-10%) and supplemented with an indicator, bromocresol purple, and incubated overnight at 37°C. Then, the growth was assessed by observing the change of the indicator from purple to yellow.

Bile salt tolerance test

Isolated strains survived in acid and NaCl conditions were determined for bile salt tolerance testing as described by Reuben et al. (2019) with some modification. Briefly, overnight cultures in fresh MRS broth were centrifuged at 3,400 rpm for 10 min at 25°C. Then, resuspended the pellet in sterile phosphate buffered saline (PBS) and adjusted to 10⁸ CFU/mL, added to fresh MRS broth (pH 6.5) containing 0.3% (w/vol) bile salt (HiMedia, India). LAB culture in MRS broth without bile salt indicated as control. The culture was incubated at 37°C for 6 hours. The viability of cell was assessed by serial dilution and plating onto MRS agar at 0, 3, and 6 hours of incubation. Three separate independent measurements (triplicate) were done.

Antibiotic susceptibility test

The antibiotic susceptibility profile of the LAB strain was examined to 11 commonly used antibiotics. By using the agar disc diffusion method, the LAB strains were grown in fresh MRS broth at 37°C overnight. The suspensions were adjusted to 1.0×10⁸ CFU/mL (0.5 McFarland standard) and spread onto MRS agar. Antibiotics discs (HiMedia, India) including cefoxitin (30 µg), clindamycin (2 µg), cotrimoxazole (25 µg), ciprofloxacin (5 µg), ertapenem (10 µg), erythromycin (15 µg), penicillin (10 µg), tetracycline (30 µg), rifampin (5 µg), vancomycin (30 µg), and streptomycin (300 µg) were placed on the surfaced or inoculated agar plates, and then incubated for 24 hours at 37°C. The susceptibility test was evaluated according to the cut-off levels suggested by Charteris et al. (1998) and Suwannaphan (2021), except for Ertapenem (ETP) and Streptomycin (HLS), which were assessed according to HiMedia Laboratories guidelines as described by Duche et al. (2023) as shown in Supplement Table 1.

Antagonistic activity

To characterize for production of inhibitory substances against pathogen, the antagonistic activity was evaluated using agar diffusion method (Stern et al., 2006; Tkesheliadze et al., 2023) with some modification. The screening of antagonistic activity of LAB was tested against Staphylococcus aureus (S. aureus) ATCC 25925, Enterococcus faecalis (E. faecalis) DMST 2860, Escherichia coli (E. coli) DMST 4212 and Pseudomonas aeruginosa (P. aeruginosa) DMST 4739. Briefly, the pathogenic bacteria were culture on Mueller-Hinton agar (MHA) plates (HiMedia, India). The fresh prepared culture of each LAB strain on MRS agar was suspended in normal saline and adjusted to contain 1.0 × 10⁸ CFU/mL. Then, the suspension was spread onto MRS agar and incubated anaerobically at 37 °C for 24 hours. Agar blocks containing the growth (7 mm. in diameter) were cut and transferred to MHA containing 1.0 × 10⁸ CFU/mL of the pathogenic bacteria. The plates were incubated at 37°C for 24 hours. under aerobic conditions. The inhibition activity was evaluated by measuring the inhibition zone around the blocks which was greater than 10 mm.

Hemolytic activity

An overnight growth of LAB strain was streaked on blood agar formulated with Mueller Hinton agar (HiMedia, India) and 5% fresh human blood. Then, the appearance of hemolytic reaction after incubation (37°C, 48 hours) was examined by observing green zones surrounding colonies (α-hemolysis), clear zones surrounding colonies (β-hemolysis) and no zones surrounding colonies (γ-hemolysis).

Aggregation abilities

Auto-aggregation and co-aggregation abilities were carried out according to previous studies by Abushelaibi et al. (2017) and Reuben et al. (2019). Exactly 5 mL of the bacteria suspension with 1.0 × 10⁸ CFU/mL was vortexed for 10 seconds and measured for absorbance by spectrophotometer at 600 nm, which was recorded as the initial optical density (OD_i). Then, the suspensions were incubated for 2 hours at 37°C, and the supernatant was measured, which was recorded as OD₂. The percentage of auto-aggregation was determined as:

auto-aggregation (%) = 1- (OD₂/OD_i ) × 100

Test pathogens (S. aureus ATCC 25925, E. faecalis DMST 2860, E. coli DMST 4212, and P. aeruginosa DMST 4739) were used for co-aggregation testing. Two milliliters of each culture LAB strains and equal volume of each test pathogens were mixed. The mixed cultures were vortexed for 10 seconds and incubated for 2 hours at 37°C. The control of each pathogen culture was each bacterial suspension. At 600 nm, the absorbance for each mixed suspension was measured (OD_mix). Then, compared to those of the control of the probiotic strain as OD_strain and the tested pathogen as OD_pathogen at 2 hours of incubation. The percentage of Co-aggregation was calculated as:

Co-aggregation (%) = (1- OD_mix (OD_strain+ OD_pathogen)/2) x 100

The auto-aggregation and co-aggregation abilities of the selected LAB strains were analyzed using two-way ANOVA, followed by Tukey's multiple comparisons test.

β-galactosidase activity

The β-galactosidase activity of LAB was assessed by streaked the bacteria culture on MRS agar plates containing 60 µL of 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (X-gal) and 10 µL of Isopropyl-β-D-1-Thiogalactopyranoside (IPTG) solution. The blue colonies indicated the presence of the β-galactosidase activity as described previously (Sriphannam and Kummasook, 2020).

Characterization of LAB strain

The bacterial DNA was extracted from an overnight culture in MRS broth at 37°C using PureDireX Genomic DNA Isolation Kit (Bio-helix, Taiwan) according to the manufacturer’s instruction. The molecular identification of LAB strains was conducted by amplification of 16S rRNA gene region, using the universal forward and reverse primers F44 (5’RGTTYGATYMTGGCTCAG-3’) and R1543 (5’-GNNTACCTTKTTACGACTT-3’), respectively (Sanpa et al., 2019). A reaction mixture without a template was used as a negative control. The PCR products with 1.5 kb were detected on a 2% agarose gel with Novel Juice Dye (GeneDireX, Taiwan).

Sequence and phylogenetic analyses

The amplified PCR products were purified and sequenced, then analyzed using the Clustral X and BioEdit software programs for homology using the Basic Local Alignment Search Tool (BLAST) server (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were corrected and aligned using Clustral W, and the phylogenetic tree was constructed by MEGA11 software based on the neighbor-joining method (Srinivash et al., 2023).

Statistical Analysis

The data were shown as the mean ± standard deviation (SD) or standard error of mean (SEM) deviation after the measurements were independently performed in triplicate. Statistical analyses were performed using GraphPad Prism version 10.2.2 for Window (GraphPad Software, San Diego, CA). Statistically significant differences between means were considered at P < 0.05.

RESULTS

Twenty isolates were randomly selected based on their typical morphological appearance, including color, form margins, surface, and elevation. All these isolates were Gram-positive rod non-spore forming, catalase negative, OF test positive and able to change colour on bromocresol purple agar. All 20 strains then were tested for probiotics properties.

Probiotic properties of LAB

Survivability in acid, NaCl and bile salts conditions

All chosen LAB was tested for cell variabilities for 3 hours to assess their acid tolerance (pH 2.0). Two strains, PY13 and PY41, demonstrated tolerance to this low‑pH environment, with survival rates of 41.03% and 42.82%, respectively. Both isolates also survived in NaCl concentrations up to 7%.  According to these results, the LAB that tolerated to low pH were selected for bile salt tolerance testing. Both selected LAB showed bile salt tolerance at varying degrees ranging from 4.7 ± 0.8 to 5.1 ± 0.3 log10 CFU/mL (P > 0.05). The LAB strains were observed to have higher microbial counts after 6 hours (Figure 1). The ability to withstand 3% bile salts was assessed at 0, 3, and 6 hours. During the first 3 hours, survival rates were 95.24% for PY13 (A1‑3) and 171.43% for PY41 (A4‑1). After 6 hours, survival continued to increase, reaching 333.33% for PY13 and 664.29% for PY41.

Figure 1. Survival of LAB strains PY13 (A) and PY41 (B) in 0.3% bile salts. Results are expressed as log₁₀ CFU/mL relative to the control (0% bile salts). Data represents the mean of triplicate experiments; error bars indicate the standard error of the mean (SEM).

Antibiotic susceptibility test

The antibiotic susceptibility profile of the selected LAB strains to commonly used antibiotics was investigated (Table 1). LAB trains PY13 and PY41, were susceptible to most tested antibiotics with the exception of ciprofloxacin, and vancomycin. Additionally, PY13 and PY41 were found to be moderately susceptible to penicillin and cefoxitin.

Table 1. Antibiotic resistance profile of selected LAB.

Note: The antibiotic disc diameter is 6 mm.  The susceptibility represents as S (susceptible), MS (moderately susceptible) and R (resistant). In each column represents mean from duplicate determinations. “–” indicates no inhibition zone.

Antagonistic activity

Living organism agar block assay was performed to determine the presences of secreted antimicrobial substances. The selected strains were examined for a preliminary screening of the antagonistic activity against pathogenic bacteria, including S. aureus, E. faecalis, E. coli and P. aeruginosa.

As shown in Table 2, both strains were able to inhibit the growth of all pathogenic bacteria tested in this study, with inhibition zones between approximately 11 to 18 mm. Moreover, the results indicate that PY41 showed stronger inhibition activity against S. aureus and E. faecalis than PY13. Strain PY41 exhibited the most substantial inhibitory effect against Gram-positive bacteria, E. faecalis with an inhibition zone of 17.7 ± 0.58 mm.

Table 2. Antagonistic activity of selected LAB isolated against pathogenic bacteria.

Note: In each column represents Mean ± SD from triplicate determinations.

Hemolytic activity

Neither selected LAB strains exhibited a clear zone (α-hemolysis) nor green zone (β-hemolysis) on blood agar plates, indicating that the selected LAB strains are non-virulent strains.

Aggregation abilities

Both auto-aggregation and co-aggregation abilities of selected LAB strains were determined (Figure 2). Statistical analysis by two-way ANOVA and Tukey's multiple comparisons test were used. The auto-aggregation of PY13 and PY41 were 30.68 ± 2.53% and 28.47 ± 4.07%, respectively. The co-aggregation ability of selected strains, PY13 and PY41, with test pathogen were not significantly different with P > 0.05. Meanwhile, both selected strains showed no difference in co-aggregation ability (P > 0.05) when tested with different test pathogens.

Figure 2. Aggregation abilities of LAB selected strains, PY13 and PY41, the result represents as % aggregation. Data represent the means of triplicate experiments, and error bars indicate SEM. Auto-aggregation (A) and Co-aggregation (B) of both strains show no- significant difference (P > 0.05).

β -galactosidase activity

For the β-galactosidase screening, both PY13 and PY41 colonies were blue on MRS agar plates containing 60 µL of 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (X-gal) and 10 µL of Isopropyl- β-D-1-Thiogalactopyranoside (IPTG) solution. This indicates that these bacteria have β -galactosidase activity.

Molecular characterization of LAB strain

The selected LAB strains were identified by 16S rRNA amplification and sequencing. Subsequently, these strains were blasted and deposited in GenBank database under the accession numbers (PP380006-PP380007). BLAST analysis of PY13 and PY14 sequences indicated that isolates corresponded LAB with genetic similarity values close to 100% with those reported in GenBank (Table 4). Molecular phylogenetic analysis and phylogenetic tree were performed to identify LAB strains based on the 16S rRNA sequences using evolutionary distances by neighbor-joining method (Figure 3). Both strains showed 100% nucleotide sequence identity as Lactiplantibacillus pentosus (formerly Lactobacillus pentosus; Zheng et al., 2020).

Table 3. BLAST identification of LAB isolated from Pla-som product.

Figure 3. Phylogenetic tree analysis of LAB isolated from Pla-som by neighbor-joining method.

DISCUSSION

The manufacturing process of traditional Thai fermented fish, pla-som, depends on a spontaneous fermentation that is started by naturally occurring microorganisms, primarily LAB, which are present in the materials, on the processing equipment, and in the surrounding air as natural starters (Kopermsub and Yunchalard, 2010). LAB can improve immunological function and digestion (Behnsen et al., 2013). LAB are recognised as probiotics, living microorganisms that are known to be natural alternatives to antibiotics and offer health-promoting benefits to the host by improving immunological function and digestion (Behnsen et al., 2013). Through modulation of the immune system, LAB increases the efficiency of nutrient utilisation and eliminates the presence of pathogens. According to Rabetafika et al. (2023), probiotics serve as a promising alternative to antibiotics, addressing the global challenge of antimicrobial resistance (AMR) by restoring microbial balance rather than merely treating infections.

In the present study, 20 LAB strains were isolated from Pla-som from Phayao province, Thailand. The probiotic properties of the isolated strains were determined for their safety and health benefits. The potential probiotics LAB is suggested to survive in the gastrointestinal tract, guaranteeing their viability and functionality. Due to the secretion of gastric juices, the stomach condition has a low pH (1.5-3.0) (Fontana et al., 2013; Srinivash et al., 2023). The results from this study indicate that both selected strains could tolerate pH 2.0 for 3 hours. These are in good agreement with the previous studies which demonstrate that LAB strains could remain variable when incubated at pH 2.0 (Song et al., 2015; Reuben et al., 2019; Sriphannam and Kummasook, 2020; Srinivash et al., 2023). While our model focused on the impact of gastric acidity, we acknowledge that the presence of pepsin in vivo may further influence LAB survival (Corcoran et al., 2005). NaCl is an important factor for LAB to grow in fermented condition and is used to inhibit the growth of pathogenic and spoilage bacteria in fermented food (Bremer, 2000). As described by the Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, 2002, gastric survival in vivo has been demonstrated to correlate with in vitro bile salt resistance. In this study, both selected strains were resistant to gastric acidity and were able to survive and multiply at 0.3% bile salt after 6 hours of incubation. The results corresponded to the findings of Sriphannam and Kummasook (2020), in which LAB strain isolated from a similar source, fermented fish (Pla-som), showed resistance to 0.3% bile concentration for 24 hours incubation. Moreover, these results concur with previous studies that LAB isolated from raw milk and Kashar cheese (traditional cheese from Turkey) were also tolerant to bile salts at 0.3% (Tang et al., 2018; Reuben et al., 2019; Ertürkmen et al., 2024).

Antibiotic susceptibility, or lack of antibiotic resistance, is an important safety factor for probiotic strains (Srinivash et al., 2023). Probiotic strains should not carry antibiotic resistance genes as a reservoir, which could be transferred to intestinal pathogens (Nallala et al., 2017; Reuben et al., 2019). In this study, both selected strains showed resistance to vancomycin and ciprofloxacin. LAB has been reported to have intrinsic resistance to vancomycin, which is a tricyclic glycopeptide antibiotic due to the cell wall synthesis inhibitor (Patel et al., 2024). Various studies report resistance to vancomycin by LAB isolated from different products. Natural resistance of LAB to vancomycin was documented by Erginkaya et al. (2018) which investigated the antibiotic resistance of LAB isolated from traditional Turkish fermented dairy products and reported a high level of vancomycin resistance. Due to low affinity binding to altered peptidoglycan termini of vancomycin, LAB can resist vancomycin (Anisimova and Yarullina, 2019). Various strains of Lactobacillus spp., a type of LAB, demonstrated vanX, a crucial gene for vancomycin resistance. This gene produces D-alanyl-D-alanine dipeptidase which prevents the synthesis of usual D-alanyl-D-alanine termini of the peptidoglycan precursor side chain. Furthermore, resistance to quinolone antibiotic, including ciprofloxacin has been reported by several studies (Sharma et al., 2016; Anisimova and Yarullina, 2019; Duche et al., 2023). This may be due to intrinsic resistance mechanisms such as cell wall impermeability of efflux mechanism. However, some of the intrinsic resistance mechanisms may result from mutations involving genes such as parC (topoisomerase IV), the quinolone resistance-determining region (QRDR), and gyrA (DNA gyrase) (Anisimova and Yarullina, 2019).

According to FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, 2002, bacterial strains to be used as probiotics are recommended to be tested to have antimicrobial activity against pathogens testing for safety considerations. The antagonistic activity was necessary to test for use as a starter culture as they can prevent food spoilage and inhibit the growth of pathogenic bacteria (Hwanhlem et al., 2011). LAB was able to produce antibacterial compounds during growth, including metabolites, organic acids, and bacteriocins (Abushelaibi et al., 2017). Our results concur with Reuben et al. (2019), who reported the antimicrobial activity of LAB isolated from raw cow and goat milk against an array of pathogenic bacteria with a different method, agar well diffusion assay. Moreover, Hwanhlem et al. (2011) observed the antagonistic activity of 14 LAB strains isolated from the similar product toward S. aureus and E. coli. Gram-negative bacteria (E. coli and P. aeruginosa) have an additional outer membrane which presence as lipopolysaccharide (LPS) layer, whereas Gram-positive bacteria (S. aureus and E. faecalis) have a thick cell wall. Depending on LAB's ability to reduce intracellular pH and disrupt the transmembrane proton motive force, LAB is able to disrupt the LPS layer in Gram-negative bacteria. These abilities also help the undissociated form of molecules such as bacteriocin, enzymes or hydrophobic substances to access the periplasm (Hwanhlem et al., 2011).

No type of haemolysis on human blood agar, α-hemolysis or β-hemolysis, was exhibited by the selected LAB strains. The absence of hemolytic activity is considered to be safety requirement for selecting a probiotic strain. Previous reports indicated similar results that most LAB strains have no zone of haemolysis or γ-hemolysis (Reuben et al., 2019; Srinivash et al., 2023). The lack of hemolytic activity is a safety consideration when determining a probiotic strain (Ahire et al., 2021).

Auto-aggregation and co-aggregation were examined for the potential probiotic strains cell binding properties. Auto-aggregation is considered as aggregation between the same microbial strains. This correlates with the bacterial adhesion to epithelial cells of the gastrointestinal, similarly to previous studies that reported the auto-aggregation of LAB (Sampaio et al., 2021; Srinivash et al., 2023). However, auto-aggregation of potential probiotic strains higher than 40% is required (Roghmann and McGrail, 2006). Our results indicated auto-aggregation levels were 30.68 ± 2.53% and 28.47 ± 4.07%, which were below 40%. This suggests that the selected strains depend less on self-binding and more on certain surface proteins, such as Mucus-Binding Proteins (Mubs), to attach to the host's intestinal wall. Co-aggregation is defined as the aggregation between different microbial strains. Potential probiotics can co-aggregate with pathogens, suppress their growth, and then kill them with their antimicrobial compounds (Riaz Rajoka et al., 2017). Moreover, auto-aggregation and co-aggregation may determine the capacity of probiotic bacteria to form biofilms that protect hosts and inhibit disease invasion (Khalil et al., 2018). In contrast to the auto-aggregation data, in this study, the selected strains have exhibited strong co-aggregation level, exceeding 40%. Therefore, our strains may be crucial in assisting the gastrointestinal tract in eliminating pathogens.

The results indicate that β-galactosidase can be produced by the selected LAB strains. This enzyme is the major factor for improved digestibility by hydrolysis of β-glycosidic bonds between galactose and glucose (Kolev et al., 2022). In addition, the enzyme can catalyse the trans-galactosylaion of lactose. Several studies indicate that LAB is able to produce this enzyme which possesses probiotic properties. A deficiency of this enzyme in the human intestine leads to lactose intolerance. Thus, people suffering from it have problems from consuming milk or dairy products (Saqib et al., 2017).

Both LAB strains identified in this study were identified as Lactiplantibacillus pentosus (formerly Lactobacillus pentosus (Song et al., 2015)) with 100% identity. This identified bacterium has been reported as a probiotic strain isolated from different sources, including fermented table olives (Abriouel et al., 2022; Zotta et al., 2022). Consistent with previous reports identifying Lactiplantibacillus pentosus in Pla-som (Sanpa et al., 2019), our findings further confirm the presence of this species in this traditional fermented fish. Moreover, the specific strain isolated in this study demonstrates robust characteristics, suggesting it as a promising candidate for a potential probiotic strain. Lactococcus garvieae, Streptococcus bovis, Weissella cibaria, Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum have been identified from Pla-som (Kopermsub and Yunchalard, 2010; Sanpa et al., 2019). Both strains, PY13 and PY41, can be developed as starter microorganisms in food products and dietary supplements because of their safety and were identified as unharmful species for human consumption. These chosen microorganisms can survive harsh gastrointestinal conditions, including acidic stomach pH and bile salts, to reach the intestines intact, demonstrate antimicrobial activity and inhibit pathogenic microorganisms. Furthermore, both strains need for more standardized practices in the industry. These would be beneficial to develop the Pla-som products to the industry level.

CONCLUSION

According to our results, two potential LAB strains were isolated from Pla-som, PY13 and PY41. These strains showed good probiotic potential including acidic, NaCl and bile salts tolerant, β-galactosidase production, aggregation abilities, non-hemolysis strain, and antagonistic activity. Moreover, both strains were susceptible to most antibiotics except for ciprofloxacin. Through 16S rRNA sequencing, PY13 and PY41 were identified as Lactiplantibacillus pentosus. The study's results indicate that these isolated LAB strains have good probiotic potential and could serve as starter microorganisms in food products and dietary supplements. Further study is required to confirm the safety of those strains for other health benefits such as adherence to mucus and/or human epithelial cells and cell line, exopolysaccharide production, lysozyme tolerance and cholesterol removal. 

ACKNOWLEDGEMENT

The authors would like to thank the Department of Medical Technology, School of Allied Health Sciences, University of Phayao for providing laboratory facilities.

AUTHOR CONTRIBUTIONS

Kanittapon Supadej: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Validation (Lead), Resource (Equal), Data Curation (Lead), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal), Visualization (Equal), Supervision (Lead), Project Administration (Lead); Natnicha Khamfan: Investigation (Supporting), Data Curation (Equal), Formal Analysis (Supporting), Writing – Original Draft (Equal); Boonyika Doosorn: Investigation (Supporting), Data Curation (Equal), Formal Analysis (Supporting), Writing – Original Draft (Equal); Philaiphon Jongruamklang: Formal Analysis (Supporting), Writing – Review & Editing (Equal), Validation (Equal), Visualization (Supporting); Aksarakorn Kummasook: Conceptualization (Supporting), Methodology (Lead), Resource (Equal), Supervision (Supporting), Project Administration (Supporting); Benjamart Suradej: Conceptualization (Lead), Resource (Equal), Formal Analysis (Lead), Validation (Equal), Writing – Review & Editing (Equal), Project Administration (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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