Characterization of lactic acid bacteria from Nono, a Nigerian naturally fermented milk product

Obioha, Promiselynda Ijeoma (2019) Characterization of lactic acid bacteria from Nono, a Nigerian naturally fermented milk product. Doctoral thesis, London Metropolitan University.


The aim of this research was to enumerate, isolate, identify and characterize lactic acid bacteria (LAB) from Nono, a Nigerian naturally fermented cow milk product for the selection of multifunctional starter cultures to develop a controlled fermentation process for Nono production. This study focused on screening the diversity of the LAB associated with Nono and characterizing their potential probiotic and functional properties including tolerance to acidic pH and bile salt, antimicrobial activity against indicators of food borne pathogens, and resistance to antimicrobials.
The LAB were enumerated and isolated from Nono using MRS, M l7 and MRS + LCysteine agars. These media were selected to harvest a diverse range of LAB associated with Nono. Further, they were identified using conventional phenotypic methods including Gram, catalase and oxidase and the Standard Analytical Profile Index (API 50 CHL) identification system. Genotypic methods including repetitive element sequence-based polymerase chain reaction (rep-PCR) and sequencing of the 16S rRNA, phenylalanyl-tRNA synthase a-subunit (pheS) and RNA polymerase, alpha subunit (rpoA) genes were used to identify the bacteria. The sequences were analysed using the Genbank and Eztaxon databases. Phenotyping revealed a bacterial count at a level of 107 CFU/ml for all samples. A total of 128 LAB were isolated and characterized as Gram positive, catalase and oxidase negative and non-spore forming bacteria. The shape and organization of the isolates were variable: rod, cocci, vibrio, V-shaped and coccobacillus bacteria organized as single, pairs or groups. The repPCR allowed the differentiation of multiple groups within the same species and sequencing of the 16S rRNA, pheS and rpoA genes allowed the identification of various genus and species including Lactobacillus fermentum (40%), Lactobacillus senioris (2%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%) Streptococcus infantarius (10%), Leuconostoc pseudomesenteriodes (2 %) and Enterococcus thailandicus (1%>).
Further characterisation of the isolates for probiotic and functional properties focused on seven isolates selected on the basis of differences in their rep-PCR profiles. These include Lactobacillus fermentimi, Lactobacillus senioris, Lactobacillus delbrueckii, Streptococcus thermophilus, Streptococcus infantarius, Leuconostoc pseudomesenteriodes, and Enterococcus thailandicus. The isolates were screened for tolerance to different acidic pH and bile salt concentrations to characterise their resistance to gastric acid and bile. The survival of the isolates at different acidic pH varied according to the isolates and incubation time, Lactobacillus fermentum followed by Lactobacillus senioris survived better at pH 3 and pH 4 for 3 h incubation compared to other test isolates. All the isolates survived high concentration 1.5% and 2% of the bile salt for 3 h incubation. The isolates were further screened for antimicrobial activities against indicators of pathogenic bacteria including Samonella enteritidis serovar Typhimurium variant D TI24, Escherichia coli NCTC 12900, Listeria monocytogenes NCTC 11994, Staphylococcus aureus CMCC 1930 and Bacillus cereus LMG 1356. Inhibitory activity of the test organisms was evaluated using a spot test and also spectrophotometric method by measuring and comparing the optical density (OD) of the indicator bacteria after the 24 h incubation period in both test and control experiment. The test isolates exhibited varying levels of inhibition against common Gram positive and Gram negative foodbome pathogens. Among the seven species of LAB screened for antimicrobial activity, Lactobacillus fermentum not only showed broad antimicrobial activities against the indicators but also exhibited antilisterial activity against Listeria monocytogenes and this is of significant spotlight in the starter culture selection. The susceptibility of the selected LAB to 18 antimicrobials was evaluated by screening the Minimal Inhibitory Concentration (MIC) for each antimicrobial.
This was followed by the detection of resistance genes by PCR. The ability of two isolates of LAB to transfer to other bacteria the tet(S), tet{M) genes coding for tetracycline resistance and aad(E) gene coding for streptomycin resistance was investigated by conjugation experiments.
The latter experiements revealed a variable antimicrobial susceptibility according to the LAB isolate and the antimicrobial tested. The tet(S), tet(M) were detected in the isolates of Enterococcus thailandicus (52) and Streptococcus infantarius(lO).
Additionally, aad{E) was detected in Enterococcus thailandicus (52). The conjugation experiments suggested that the tet(S) gene was transferable in vitro from isolates 52 and 10 to E.faecalis JH2-2 and aad(E) from 52 only to E.faecalis JH2-2.
Both tet(S) and aad(E) are located at least on plasmids that have mediated the transfer of the genes to Enterococcus faecalis JH2-2 because positive amplicons were obtained in the donors and transconjugants by amplification of the gene from plasmid DNA samples.
This research concluded that various genus, species and sub-species of LAB are involved in the production of Nono. The data obtained in this research are relevant for the selection of multifunctional starter cultures for a control production of Nono in Nigeria.

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