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Isolation, evaluation, and whole genome analysis of lactic acid bacteria and their application as starter culture for Lao traditional fermented bamboo shoots (Nor mai som)
Dissertation Abstract:
A total of 20 lactic acid bacteria (LAB) were isolated from Nor mai som. Based on initial screening, five isolates, 1, 3, 13, 16, and 20, were selected for species identification by 16S rRNA gene sequence analysis and were found to be Pediococcus pentosaceus BBS1, Lactobacillus pentosus BBS3, Lactiplantibacillus plantarum BBS13, Lactiplantibacillus plantarum BBS16, and Lactobacillus pentosus BBS20. All of them showed antimicrobial activity and were positive for proteolytic and linamarase activity. They were able to grow in De Man, Rogosa and Sharpe (MRS) and in screening media containing potassium cyanide (KCN) solution and remained viable after exposure to highly acidic pH (2.0) and bile salt at concentration of 0.3%, for 3 h at 37°C. They did not show hemolytic activity and were sensitive to various antibiotics.
In-vitro tests for the evaluation of the probiotic properties of strains BBS1 and BBS13 revealed their tolerance to simulated gastrointestinal tract conditions. Bacterial adhesion to xylene and chloroform was obtained for BBS1 (36.88% and 40.17%) and BBS13 (39.54% and 49.33%), respectively. The highest scavenging activity was found in BBS13 at 77.41% and BBS1 at 72.74%.
Whole genome sequencing (WGS) analysis and safety assessment were done for strains BBS1 and BBS13. Species identification by average nucleotide identity (ANI) gave the following results: Pediococcus pentosaceus BBS1 (99.60%) and Lactiplantibacillus plantarum BBS13 (99.5%). The circular chromosome was composed of 1,840,613 bp and 3,238,769 bp for strains BBS1 and BBS13, respectively. The two strains contained Lacetate dehydrogenase and D-lactate dehydrogenase, the genes responsible for lactic acid production, as well as linamarase or B-glucosidases. Both strains had no virulence factors, biogenic amines, or antimicrobial resistance genes, thereby eliminating safety concerns.
Strains BBS1 and BBS13, when applied to the Nor mai som fermentation, were able to decrease the pH values to approximately 3.0, which consequently raised the lactic acid levels in all the treatments. All Nor mai som samples had >9 log CFU/g for LAB and total viable count (TVC) populations at 24 h, and declined thereafter. Enterobacteriaceae were undetectable in all samples with inoculated starter cultures, whereas spontaneous fermentation still had 3.95 log CFU/g on day 3. On day 10, treatments with inoculated strains showed significant cyanide reduction, ranging from 62.09-90.31% compared to natural fermentation at 49.75%. These results suggest that the LAB strains from this study could be employed as probiotic starter cultures to improve the quality of Nor mai som.
