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

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

Antimicrobial resistance of lactic acid bacteria from Nono, a naturally fermented milk product

Background: Antimicrobial resistance (AMR) is one of the biggest threats to public health. The food chain has been recognised as a vehicle for transmitting AMR bacteria. However, information about resistant strains isolated from African traditional fermented foods remains limited. Nono is a traditional, naturally fermented milk product consumed by many pastoral communities across West Africa. The main aim of this study was to investigate and determine the AMR patterns of lactic acid bacteria (LAB) involved in the traditional fermentation of milk for Nono production, and the presence of transferable AMR determinants. Methods: One hundred (100) LAB isolates from Nono identified in a previous study as Limosilactobacillus fermentum, Lactobacillus delbrueckii, Streptococcus thermophilus, Streptococcus infantarius, Lentilactobacillus senioris, Leuconostoc pseudomesenteriodes, and Enterococcus thailandicus were investigated. The minimum inhibitory concentration (MIC) was determined for 18 antimicrobials using the micro-broth dilution method. In addition, LAB isolates were screened for 28 antimicrobial resistance genes using PCR. The ability of LAB isolates to transfer tetracycline and streptomycin resistance genes to Enterococcus faecalis was also investigated. Results: The experiments revealed variable antimicrobial susceptibility according to the LAB isolate and the antimicrobial tested. The tetracycline resistance genes tet(S) and tet(M) were detected in isolates Ent. thailandicus 52 and S. infantarius 10. Additionally, aad(E) encoding resistance to streptomycin was detected in Ent. thailandicus 52. The conjugation experiments suggested that the tet(S) and aad(E) genes were transferable in vitro from isolate Ent. thailandicus 52 to Ent. faecalis JH2-2. Significance and Impact: Traditional fermented foods play a significant role in the diet of millions of people in Africa, yet their contribution to the burden of AMR is largely unknown. This study highlights that LAB involved in traditionally fermented foods could be potential reservoirs of AMR. It also underscores the relevant safety issues of Ent. thailandicus 52 and S. infantarius 10 for use as starter cultures as they carry transferable AMR genes. Starter cultures are an essential aspect of improving the safety and quality attributes of African fermented foods. However, AMR monitoring is an important safety aspect in the selection of starter cultures for improving traditional fermentation technologies

Identification and characterisation of the lactic acid bacteria associated with the traditional fermentation of dairy fermented product

The aim of this research was to identify the key lactic acid bacteria associated with the fermentation of dairy traditional fermented products for developing starter cultures for controlled fermentation. A total of 100 lactic acid bacteria (LAB) were isolated from dairy traditional fermented products. Samples were obtained from eight producers in the South East of Nigeria. Isolates were identified by phenotypic and genotypic techniques including rep-PCR genotyping and sequencing of the 16S rRNA, pheS and rpoA genes. Isolates were characterised for antimicrobial activity against foodborne pathogens, exopolysaccharide (EPS) production and survival at low pH and in the presence of bile salts. All isolates clustered into 11 distinct rep-PCR groups and were identified as Lactobacillus fermentum (40%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%), Streptococcus infantarius (10%), Lactobacillus senioris (2%), Leuconostoc pseudomesenteriodes (2%) and Enterococcus thailandicus (1%). Lactobacillus fermentum showed a broad spectrum antimicrobial activity and survival at low pH, while Lactobacillus delbrueckii was able to tolerate low pH and produce EPS. All isolates survived in vitro exposure to 1% (w/v) bile salts over a 3-h period. L. fermentum, L. delbrueckii and S. thermophilus could be used to simulate the fermentation of dairy traditional fermented products