Isolation and characterization of lactic acid bacteria from traditional fermented bamboo shoot

Introduction

About 1.4 billion tonnes of bamboo biomass is harvested annually worldwide, and a sizable fraction of the young edible shoots undergoes spontaneous lactic acid fermentation in communities across South and Southeast Asia [1] . Fermented bamboo shoot products known variously as naw mai dong, soibum, mesu, and lung-siej serve as flavour enhancers, preservatives, and dietary fibre sources in regions where refrigeration is scarce [2] . The fermentation is driven by indigenous LAB that convert sugars into lactic acid, lowering the pH to 3.5-4.0 and creating conditions unfavourable to spoilage organisms and pathogens [3] . Interest in these traditional products has grown for two reasons. First, consumer demand for naturally fermented and probiotic-rich foods has expanded beyond yogurt and kimchi into less-explored substrates [4] . Second, LAB from unconventional niches sometimes carry unique metabolic traits broad-spectrum bacteriocin production, exopolysaccharide synthesis, or resistance to harsh gastrointestinal conditions that dairy-origin strains lack [5] . Tamang et al. [6] reported that fermented bamboo products from northeastern India harboured L. plantarum, L. brevis, and Leuconostoc mesenteroides, but the species composition shifted with geography and preparation method. In northern Thailand, bamboo fermentation follows a simpler protocol than in the northern highland regions: fresh shoots are sliced, salted lightly, packed in earthen pots, and left at ambient temperature for 7-15 days [7] . Despite widespread household use, the microbial ecology of this regional variant hasn't been mapped, and no isolates from this region have been tested for probiotic potential. This research therefore aimed to isolate LAB from traditionally fermented bamboo shoot collected in Chiang Mai province, identify species by molecular methods, and evaluate selected strains for acid tolerance, bile resistance, and antimicrobial activity.

Methods

Ten grams of each fermented sample were homogenized in 90 mL sterile peptone water, serially diluted (10⁻¹ to 10⁻⁷), and plated on MRS agar supplemented with 0.5% CaCO₃.

Plates were incubated anaerobically at 37 °C for 48 h.

Colonies showing clear zones (acid production) were picked, purified by streaking three times, and stored at -20 °C in MRS broth with 20% glycerol. Gram staining, catalase testing, and carbohydrate fermentation profiles (API 50 CHL, bioMérieux) provided preliminary identification. Molecular confirmation used 16S rRNA gene amplification with primers 27F and 1492R [8] , followed by Sanger sequencing and BLAST comparison against the NCBI GenBank database (≥98% identity threshold). Acid tolerance was assessed by incubating log-phase cultures in MRS broth adjusted to pH 2.5 and 3.0 with HCl for 3 h at 37 °C; viable counts before and after exposure gave survival rates [9] . Bile tolerance followed a similar protocol using 0.3% oxgall in MRS broth for 4 h [10] . Antimicrobial activity was measured by the agar well-diffusion method: cell-free supernatant (100 µL) was placed in 8 mm wells on Mueller-Hinton agar seeded with each pathogen, and inhibition zone diameters were recorded after 24 h [11] . All tests were performed in triplicate. Data were analysed by one-way ANOVA and Duncan's multiple range test at p < 0.05 using SPSS v26. From 87 initial isolates, 23 morphologically distinct colonies were selected; all were Gram-positive, catalasenegative rods or cocci. Sequencing assigned them to six species (Figure 1). L. plantarum dominated at 34.7%, followed by L. brevis (22.1%) and L. fermentum (17.8%).

Results

Pediococcus pentosaceus accounted for 14.3%, while L. casei and minor unidentified taxa together made up 11.1%. Excluded after morphology Classification based on thresholds adapted from FAO/WHO guidelines for probiotic evaluation [12] The screening funnelled 87 raw isolates down to two strong probiotic candidates. LP-1 and LB-1 stood out because they combined high acid survival (>78% at pH 2.5), strong bile tolerance (>74% at 0.3% oxgall), and broad antimicrobial reach (inhibition zones >14 mm against both Gram-positive and Gram-negative pathogens). The fermentation kinetics trial with LP-1 confirmed rapid acidification, dropping the pH from 6.4 to below 4.0 within 24 h, which aligns with its dominance in the natural fermentation. Species like P. pentosaceus and L. casei, while common in fermented bamboo from other regions, showed weaker gastrointestinal survival here.

Discussion

The main finding of this research is that traditional fermented bamboo shoot from the Chiang Mai region of International Journal of Agriculture and Food Science https://www.agriculturaljournals.com northern Thailand hosts a diverse LAB community led by L. plantarum and L. brevis, with at least two strains meeting the basic safety and functional criteria for probiotic use. This species profile agrees broadly with earlier surveys from the other Southeast Asian countries. Tamang et al. [6] identified L. plantarum as the dominant species in soibum from northeastern India, and Romi et al. [13] reported L. brevis alongside L. plantarum in Meghalaya bamboo ferments in India. The agreement across geographically distant regions suggests that bamboo shoot chemistry selects for these species regardless of local inoculum.

Where the present results differ is in the relative abundance of P. pentosaceus 14.3% here, compared with less than 5% in Southeast Asian products [6] . One possible explanation is the higher ambient temperature in central Thailand (32-38 °C during the fermentation period) versus the cooler northeast (18-28 °C), as P. pentosaceus grows faster at elevated temperatures [14] . Another possibility is the lighter salting practice in the Chiang Mai area, which may relax osmotic stress and allow cocci to compete more effectively against rod-shaped lactobacilli.

The acid and bile tolerance values for LP-1 (82.4% survival at pH 2.5, 79.6% at 0.3% bile) compare well with commercial probiotic benchmarks; Lactobacillus rhamnosus GG, for instance, retains about 80-85% viability under similar conditions [15] . Antimicrobial activity against L. monocytogenes (12.1 mm for LP-1) is particularly relevant for food safety applications, since Listeria contamination in ready-to-eat foods remains a persistent concern [16] . But these are in vitro results, and translating them into real-food matrices requires controlled feeding trials and stability testing in product formulations steps beyond the scope of this work [17] .

Conclusion

This research contributes the first molecular-level characterisation of LAB from traditionally fermented bamboo shoot in the Chiang Mai region of Thailand. The identification of L. plantarum LP-1 and L. brevis LB-1 as strains with combined acid tolerance, bile resistance, and antimicrobial activity represents a step toward developing region-specific probiotic cultures. Their ability to lower pH below 4.0 within 24 h and inhibit common pathogens supports their potential as starter cultures for controlled bamboo shoot fermentation, which could improve safety and consistency over current household practices. For the broader field of functional foods, these isolates widen the pool of candidate probiotics beyond dairy-origin strains.

Open questions remain about their survival in actual food matrices, long-term genetic stability under industrial conditions, and whether their antimicrobial compounds are bacteriocins or simply organic acids answers that will determine whether LP-1 and LB-1 move from laboratory curiosity to practical application.

Acknowledgement

Acknowledgements Funding sourcesThis work was funded through the departmental research allocation of Chiang Mai University. Institutional supportThe authors acknowledge the Department of Food Science and Technology, Chiang Mai University, for providing microbiological laboratory infrastructure and anaerobic culture facilities.

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