Characterization of novel probiotic lactic acid bacteria isolated from Malaysian fermented shrimp (Pekasam senek) with cholesterol-lowering effect / Dayang Hanim Alysha Abang Hamdani

Lactic acid bacteria (LAB) from fermented foods are proven to be able to hinder the growth and activities of some foodborne pathogens. This study aims to isolate and characterize the potential LAB isolated from Malaysian fermented shrimps, Pekasam senek. Twenty potential LAB isolates were assessed for their antagonistic activities against Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603 and Shigella sonnei ATCC 11778 via the agar spot antimicrobial assay. The antibiotic susceptibility patterns of the isolates to six types of antibiotics were assessed through the disc diffusion method. The potential isolates then were proceeded with a subsequent assay, which is the hemolytic assay where the fresh bacterial cultures were streaked on a fresh blood agar plate containing 5% (w/v) sheep blood, and incubated at 37°C for 24-48 hours to examined for signs of β-hemolysis, αhemolysis and γ-hemolysis. All selected isolates were further evaluated for their acid and bile salt tolerance. Moreover, the potential LAB isolates were characterized using 16S rRNA genes and were continued with evaluating cholesterol-lowering activity. In antagonistic evaluation, the results showed that all LAB isolates could inhibit the growth of indicator bacteria with the highest inhibition zone (31 mm) produced by PS26 against E. coli. Furthermore, nineteen (19) isolates were susceptible to ampicillin, bacitracin, chloramphenicol, and erythromycin whilst resistant to streptomycin. Twelve (12) potential isolates showed negative hemolytic activity and were preceeded for evaluating acid and bile salt tolerance. The potential isolates showed satisfactory tolerance in harsh acidic condition of pH 3.0; ten potential isolates (PS13, PS14, PS16, PS17, PS20, PS24, PS26, PS27, PS31, PS33) exhibit over 90% of tolerance, isolate PS22 with 85.37% of tolerance, and isolate PS15 with 75.73% of acid tolerance. While in bile salts tolerance, nine potential isolates (PS13, PS14, PS15, PS16, PS17, PS24, PS27, PS31, and PS33) survived with 50% to 95% survivability rate after 6 hr of exposure. Despite showing a low tolerance to bile salt compared to the control, isolates PS20, PS22, and PS27 were still growing. The strains were identified as Lactiplantibacillus plantarum (PS13, PS15, PS17, PS20, PS24, and PS31), Bacillus cereus (PS14, and PS16) and Bacillus spp. (PS22, PS26, and PS27) using 16S rRNA gene sequencing. Lastly, PS24 and PS31 showed the highest cholesterol lowering with 80.50% and 80.19% respectively. While PS13, PS14, PS16, PS17, PS20, PS26, PS27, and PS33 showed cholesterol lowering above 70%, whilst PS22 showed only 50.55% reduction. This study indicates that LAB isolated from Pekasam senek had significant antagonism ability against the tested indicator bacteria with negative hemolysis. Meanwhile, the antibiotic susceptibility, acid and bile salt tolerance patterns of the isolates were varied depending on different types of antibiotics, pH, and bile salt concentration. These results proved that the isolates have good properties as probiotics with positive cholesterol lowering activity which can promote optimal health and minimize the illness risk.