Probiotic, starter culture and antimicrobal properties of lactic acid bacteria isolated from Ugandan traditional ghee

Abstract

Ghee is one of the major fermented foods consumed in Uganda despite the fact that it is largely produced traditionally raising issues of quality control and assurance. This study investigated the bacterial diversity of traditional ghee obtained from different regions of Uganda in an effort to determine the technological properties of its dominant lactic acid bacterial (LAB) flora. A total of nine samples were purchased in original packaging, from Buganda (2), Bunyoro (2), Teso (2) & Ankole (2) regions including a control sample and subjected to microbial analysis using conventional and molecular protocols to obtain an overview of the microbial diversity of these different ghee varieties in order to focus on the dominant LAB flora. Traditional ghee from Teso was contaminated with coliforms and E. coli, but the latter were not isolated which could be due to failure to collect these organisms from random colonies selected from the media plates during isolation. The species identified using 16S rDNA sequence analysis include Enterococcus faecium (22 isolates, 30%), Lactobacillus plantarum (20 isolates, 28%), Lactobacillus rhamnosus (11 isolates, 15%), Enterococcus hirae (11 isolates, 15%), Enterococcus faecalis (2 isolates, 3%) and Bacillus cereus (2 isolates, 3%), with Lactobacillus spp. being the dominant (31 isolates, 43%) genus detected in all samples. MEGA X analysis of the 16S rDNA PCR gel images gave separate clusters for each of L. plantarum and L. rhamnosus; each of the clusters was heterogeneous and contained isolates from different subregions of Uganda highlighting possible genetic relationship between these Lactobacillus isolates. Since L. rhamnosus is preferred to L. plantarum for inclusion in fermented dairy products due to its probiotic properties, three (03) L. rhamnosus isolates were evaluated for potential probiotic properties, antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Salmonella enterica, and growth and acid production profiles in milk, in order to establish their prospects for application as probiotics, bio-preservatives and/or starter cultures in milk and other fermented foods. Each of the tested isolates exhibited good acid tolerance at the exposure of 0.7% ox bile at 30ºC for 0, 3, 6 and 9 h and remained viable (3 log reduction) after 3 h of exposure at pH 3, did not produce biogenic amines and had high level of auto-aggregation at 24 h. However, all the isolates were sensitive to bile salts implying that they may not survive the xii entire gut transit. On agar overlay assays, each of the L. rhamnosus isolates had antimicrobial activity against at all the tested indicator bacteria at 30oC, 24 h and 37oC, 24 h for LAB isolates and indicator bacterial strains, respectively. In the disc diffusion assays, only neutralized cellfree supernatants for each of the Lactobacillus isolates lost antimicrobial activity, implying that activity was due to acid production. Subsequently, two L. rhamnosus isolates were studied further for growth kinetics and acid production prospects in UHT milk model to determine their starter culture potential. All the L. rhamnosus isolates had similar growth patterns in milk attaining 8-10 log cfu/ml at 48 h. However, the isolates had low acid production capacity leading to a pH decrease from 6.89-6.92 to 4.89-5.04 after 48 h of fermentation. Acid production was growth-dependent and maximum acidification occurred at exponential phase (8-24 h). It was concluded that L. rhamnosus isolated from Ugandan traditional ghee does not have prospects for application as a starter culture due to its low acidification capability. However, these organisms have some favorable probiotic and antimicrobial properties which could be exploited commercially. Further research should investigate the aroma compounds which could be produced by these organisms in milk and other fermented foods when grown alone and in coculture with L. plantarum, and/or yeasts and moulds which were also detected in the traditional Ugandan ghee. Further studies should focus on stabilization of the lactic acid bacterial isolates against bile