In Vitro assessment of the probiotic properties of lactic acid bacteria isolated from sorghum mahewu
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Fermentation of cereal grains is associated with beneficial microorganisms, referred to as probiotics. Probiotics are live microorganisms which confer health benefits to host organisms when consumed in adequate amounts. For a probiotic microorganism to exert its beneficial effects, it has to survive transit through the gastrointestinal tract (GIT) and colonise the host gut. Ability to survive simulated GIT transit in vitro, can be used to assess probiotic properties of bacteria isolated from fermented foods. The present study evaluated the in vitro probiotic potential of lactic acid bacteria (LAB) isolated from sorghum based mahewu. Nineteen LAB were isolated from different red and white sorghum mahewu batches and characterised using their biochemical properties. The LAB were then screened for ability to tolerate simulated gastric juice conditions. Each isolate was tested for ability to grow and remain viable in de Man Rogosa and Sharpe (MRS) broth adjusted to pH 2 and pH 3, MRS broth with 0.3 % bile, simulated gastric juice and for antimicrobial activity against Escherichia coli (ATCC 1129). For the tolerance to simulated gastric transit assays, cells from an overnight culture of each isolate were centrifuged at 5 000 rpm for 5 minutes, washed twice in Phosphate Buffered Saline (PBS) at pH 7.2 and inoculated into the test broth. The inoculated test broth was then incubated at 37°C for three hours. Tolerance was determined by evaluating viable counts in an aliquot of the inoculated test broth before incubation and after three hours of incubation. Growth of the isolates in the test broth was also evaluated by measuring optical density (O.D) of the culture at hourly intervals over the three hours at 620 nm using a spectrophotometer. For evaluation of antimicrobial activity of the isolates against E. coli, the culture supernatant of each isolate was used. The culture supernatant was prepared using 48 hour MRS broth cultures of each of the isolates. The 48 hour cultures were centrifuged at 10 000 rpm for ten minutes to pellet the bacteria cells. The culture supernatant of each isolate was then added to wells on Muller Hinton agar plates on which E. coli had been spread. The plates were incubated at 37°C for 24 hours. The results of the study indicated that LAB isolated from mahewu generally had good probiotic potential, 84.2 % of the isolates had all the probiotic properties tested for in the study. All the isolates remained viable after exposure to MRS broth adjusted to pH 3 for three hours at 37°C. Tolerance to pH 2 however, varied significantly (p<0.05), 89.5 % (n=19) of the isolates showed tolerance but a reduction in percentage growth compared to growth in the control MRS broth at pH 6.5. Most of the isolates, 84.7 % showed ability to tolerate MRS broth containing 0.3 % porcine bile, the rest of the isolates were not able to grow in the presence of the bile. The tolerance of the isolates to the presence of bile differed significantly (p<0.05), indicating that the mechanisms and ability to tolerate bile salts differ among LAB. All the isolates were able to inhibit growth of E.coli. The sizes of the zones of inhibition varied considerably. This was probably due to differences in the types and amounts of antimicrobial compounds produced by each isolate. Overall, the results indicated that the LAB isolates possess desirable in vitro probiotic properties. The levels of tolerance to simulated gastric conditions and antimicrobial activity of the isolates varied considerably, indicating that tolerance mechanisms might be dependent on the specific characteristics of each isolate. Generally, the different LAB isolates could be good candidates for further studies, with in vivo tests to validate their potential.
Additional Citation InformationMugwanda,K. (2015). In Vitro assessment of the probiotic properties of lactic acid bacteria isolated from sorghum mahewu (Unpublished master's thesis). University of Zimbabwe, Harare.
Subjectanalysis of variance (ANOVA)
bile salt hydrolases (BSH)
gastrointestinal tract (GIT)
tumour necrosis factor (TNF)
phosphate buffered saline (PBS)
zone of inhibition (ZOI)