Probiotic lactic acid bacteria derived from fermented Theobroma cacao fruit juice and their inhibitory effect on Gastritis causing Helicobacter pylori -

Probiotic lactic acid bacteria derived from fermented Theobroma cacao fruit juice and their inhibitory effect on Gastritis causing Helicobacter pylori

Introduction

Helicobacter pylori stands out as a prevalent bacterial culprit in global bacterial infections. Its existence within the gastric lining triggers gastritis, potentially progressing to more severe outcomes such as ulceration or the development of malignant tumors. Despite the availability of treatments, it has been observed that the eradication rate of this microorganism ranges from 65 to 80%. Treatment failures can be attributed to factors such as non-compliance with medical prescriptions, bacterial resistance, and adverse effects of antibiotics. To enhance the tolerability of treatment and increase the eradication rate of this infection, a variety of strategies are essential, with the inclusion of probiotics being noteworthy. This study concentrated on examining the inhibitory effects of a set of lactic acid bacteria (LAB) derived from fermented cocoa juice and their metabolites against various strains of H. pylori. Additionally, the assessment of the potential of these active LAB isolates as effective probiotics was undertaken.

The isolation and characterization of lactic acid bacteria (LAB)

H. pylori strains, specifically Hp0011, Hp0012, Hp0013, Hp0014, Hp0015, Hp0016, Hp00116, and HP00117, were obtained from patients undergoing gastroendoscopic examination. All H. pylori strains were cultivated under microaerophilic conditions at 37°C for 48 to 72 hours. The lactic acid bacteria used in this study were sourced from fermented cocoa juice. Cocoa pods were aseptically extracted, and the contents were placed in a sterile plastic jar for fermentation at room temperature for 48 hours.

Ten grams of the sample were combined with 90 ml of sterile phosphate-buffered saline, homogenized, and a 0.1 ml aliquot from the 10^-6 dilution was spread on MRS agar media. The plates were incubated at two distinct temperatures, 30 °C and 37 °C, for 48 hours under anaerobic conditions. Colonies obtained were sub-cultured three times to obtain individual pure colonies. Randomly selected purified single colonies were cultured in MRS broth at 37 °C for 24 hours in aerobic conditions. The subsequent steps involved morphological characterization, Gram staining, and catalase reaction for identifying the isolates. Purified lactic acid bacteria (LAB) were those that tested positive in the Gram staining technique but negative in the catalase reaction. Pellets from the MRS broth LAB cultures were re-suspended in a broth containing 15% glycerol, and aliquots were frozen for future use.

The inhibitory activity of LAB isolates

To assess antagonistic activity, we employed the agar spot-on-lawn method, where isolated LAB strains were tested against H. pylori. A 2 µl aliquot from an overnight LAB culture was applied as a spot onto MRS agar plates and incubated anaerobically at 37 °C for 48 hours. Control plates containing only MRS agar were prepared. Subsequently, H. pylori strains were overlaid onto the plates, which were then incubated aerobically at 37 °C for 24 hours. The experiment was conducted in triplicate. Following incubation, the inhibition zone surrounding the LAB spot was observed, and the results were reported as the mean inhibition zone.

Results

From fermented cocoa juice, fifty strains of bacteria were purified, and among them, 29 (58%) were identified as lactic acid bacteria (LAB). A noticeable antagonistic effect was observed in 65.52% (19/29) of the tested LAB isolates, exhibiting inhibition zones ranging from 6 to 30 mm. The highest inhibition zone (30 mm) was achieved by LAB4′, LAB6, LAB11, and LAB19 against 12.5% to 25% of the tested pathogen. The spectrum of inhibitory activity varied from 50% to 100%, with isolate BL19 demonstrating the broadest inhibitory range. Among the pathogenic strains, HP0014 was found to be the most susceptible to LAB isolates, while HP00117 exhibited the highest resistance.

LAB isolates and their Probiotic potential of active

Every examined LAB isolate exhibited a minimum of 50% cell survival at pH 2 following incubation for up to 3 hours. LAB9 and LAB11 isolates displayed resilience to acidic pH after 6 hours of incubation, and LAB12 maintained tolerance for up to 24 hours. Additionally, they showed susceptibility to antibiotics, with inhibition zone diameters ranging from 23 to 44 mm.

Conclusion

The results also indicated that certain LAB isolates released bioactive substances into the culture medium. It has been documented that in a suitable broth medium, certain LAB secrete organic acids, hydrogen peroxide, and bacteriocins to impede the growth of pathogens. Additional analysis revealed that the cell-free supernatant (CFS) from LAB 19 hinders the growth of H. pylori by producing bacteriocin, while other isolates function through the production of compounds such as organic acids.

Reference:

Kouitcheu Mabeku, L.B., Ngue, S., Bonsou Nguemo, I. and Leundji, H., 2020. Potential of selected lactic acid bacteria from Theobroma cacao fermented fruit juice and cell-free supernatants from cultures as inhibitors of Helicobacter pylori and as good probiotic. BMC research notes, 13, pp.1-7.