Microbial content and anti-microbial activity of Namibian traditionally fermented milk

[摘要] ENGLISH ABSTRACT: The incorporation of bacteriocins as biopreservatives into model food systems has been studied extensively and has been shown to be effective in the control of pathogenic and spoilage microorganisms. However, a more practical and economic option of incorporating bacteriocins into foods can be by direct addition of bacteriocin-producing cultures into food. In this study five samples of traditionally fermented Omaere was sourced from households in Namibia. The microbial consortium present was isolated and enumerated on six different selective media that included deMan, Rogosa and Sharpe Medium (MRS) supplemented with cycloheximide for lactobacilli (MRS+C), MRS supplemented with vancomycin for leuconostocs (MRS+V), MRS supplemented with ethanol for acetic acid bacteria, M17 agar for lactococci, and Chloramphenicol Glucose Agar (CGA) and Potato Dextrose Agar (PDA) for yeasts. The highest enumeration values obtained for Omaere samples 2 and 3 were from MRS+V used for the growth of Leuconostoc spp. However, for samples 1, 4 and 5 the highest values were obtained from MRS+C used for the growth of lactobacilli. This variance among samples can be attributed to the inconsistency in the preparation methods of traditionally fermented milks.After isolation and enumeration of the microbes present in each milk sample, the Harrison Disc method was used to select bacteria and yeast colonies for further testing. The primers 27F and R1492 were used to amplify a 1.5 kilobase (kb) fragment of the 16S ribosomal RNA (rRNA) gene of the selected bacteria colonies using the polymerase chain reaction (PCR). The primers ITS4 and ITS5 were used to amplify a 600 base pair fragment of the internal transcribed spacer (ITS) regions of the fungal rRNA gene and NL1 and NL4 was used to amplify the D1/D2 domain of the 26S rRNA gene of the selected yeast isolates. The resulting PCR products were sequenced and compared to sequences listed in NCBI database using the BLAST algorithm and identified according to the closest relative. The LAB found in Namibian fermented milk Omaere belonged to the genus Lactobacillus, with the predominant species Lactobacillus plantarum (52%) and in lesser numbers Lactobacillus paracasei subsp. paracasei (12%), Lactobacillus paraplantarum (8%), Lactobacillus kefiri (8%), and Lactobacillus casei (2%). The yeasts isolated were Kazachstonia unispora formerly known as Sacchromyces unisporus (9%), Saccharomyces cerevisiae (8%) and Candida pararugosa (2%).Pure cultures of the Lactobacillus spp. isolated were used to ferment milk that was inoculated with Listeria monocytogenes and Escherichia coli and their interaction was monitored over time. After 48 h of fermentation, L. monocytogenes was not detected in milk samples inoculated with Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus casei subsp. paracasei. In contrast, when Lactobacillus kefiri was inoculated with the two foodborne pathogens, after 48 h of fermentation the concentration of L. monocytogenes was reduced by 4 log and was not detected after 72 h. In milk fermented without the addition of starters, the concentration of L. monocytogenes was only reduced by 2.8 log after 72 h of fermentation. In the milk with Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus casei subsp. paracasei after 48 h of fermentation the E. coli concentration was reduced by 4 log and after 72 h of fermentation no E. coli was detected. In contrast, fermentation with Lactobacillus kefiri after 48 h resulted in a decreased concentration of 1 log and at the end of the 72 h the E. coli concentration was only reduced by 1.7 log. In milk fermented without the addition of starter the concentration of E. coli was only reduced by 1.6 log after 72 h of fermentation.The results obtained in this study show that three of the four LAB strains isolated from the Namibian fermented milk, Omaere namely, L. plantarum, L. paraplantarum, L. parcasaei subsp. paracasei had inhibitory effect against the studied foodborne pathogens. Therefore, after further characterization of the types of antibacterial agents that are produced by these LAB, they could be considered as potential candidates for development of starter cultures that can be used for the production of microbiologically safe commercial fermented milk products. However, the L. kefiri strain used in this study is likely not to be used as starter culture as it took longer to eliminate or failed to eliminate the foodborne pathogens used in the study.

[发布机构] Stellenbosch University