Characterisation and identification of the active microbial consortium present in Kepi grains

[摘要] ENGLISH ABSTRACT:Kepi is an acidic, self-carbonated milk beverage that is produced by fermentingmilk with grain-like structures that contain naturally occurring microbes, includinglactic acid bacteria (LAB) and yeasts. The specific microbes present in the Kepigrains are responsible for an acidic-alcoholic fermentation of the milk and alsocontributes to the various health properties exhibited by Kepi. The combination ofmicrobes in the Kepi grains can vary considerably depending on which type of milkis fermented, the method by which Kepi is produced, the origin of the grains andhow the grains are stored.In this study, the impact of various environmental conditions including thedifferent stages during Kepi production, grain origin, Iyophilisation and packagingin three different packaging materials, on the microbial community of Kepi grainswere studied using selective growth media, morphology and biochemicalcharacteristics. It was found that there was a general decrease in the microbialcounts from laboratory produced Kepi grains, the longer Kepi was produced on acontinuous basis. This decrease in microbial counts was also observed during thedifferent stages of Kepi production. The average LAB counts obtained fromlaboratory produced grains decreased from 1.1 x 108 cfu.q after 3 d of activationto 6.3 x 107 cfu.q' after 10 d of mass production to 9.7 x 106 cfu.q' after a further30 d of normal Kepi production. The average yeast counts increased from nodetectable yeasts after 3 d of activation to 5.7 x 107 cfu.q' after 10 d of massproduction and then decreased again to 7.2 x 106 cfu.q' after 30 d of normal Kepiproduction. The combination of the isolates varied according to the method bywhich the Kepi grains were produced and the stress conditions that were applied.Laboratory produced Kepi grains contained the following LAB: Lactobacillusfermentum, Lb. brevis 3, Lb. p/antarum, Lb. de/brueckii subsp. de/brueckii,Lactococcus /actis subsp. /actis and Leuconostoc mesenteroides subsp. cremoris.The identified yeasts and mycelial fungi were a Zygosaccharomyces strain,Cryptococcus humico/us, Candida /ambica, C. krusei, C. kefyr and Geotrichumcandidum.The influence of grain origin on the microbial content of Kepi grains wasalso investigated using samples of Kepi grains from eight different SouthernAfrican sources. The microbial counts of the various Kepi grain samples werefound to vary from 6.0 x 105 cfu.q to 1.7 x 108 cfu.q. Five Lactobacillus, twoLeuconostoc, four Candida, one Saccharomyces and a Zygosaccharomyces strainwere isolated from these grains, with each grain type having its own uniquemicrobial combination.The microbial content of the Kepi grains that were Iyophilised,packaged in three different packaging materials and stored at room temperaturefor two months, was very similar. Lactobacillus delbrueckii subsp. delbrueckii wasisolated from the Kepi grains packaged in low density polyethylene film (LOPE).The grains packaged in oriented polyester film (OPET) contained Lb. delbrueckiisubsp. delbrueckii and Lb. brevis, while Lb. delbrueckii subsp. delbrueckii and Lb.curvatus was present in the grains packaged in methallised oriented polyesterfilm (MOPET). The average microbial counts obtained from the Kepi grainspackaged in OPET (2.7 x 106 cfu.q') were only slightly higher than that of thegrains packaged in LOPE (1.2 x 106 cfu.q') and OPET (1.4 x 106 cfu.q'). It wasconcluded that packaging materials for Kepi grains should rather be evaluated onthe quality of Kepi produced with the packaged grains than by the specificcharacteristics of the packaging materials.The enrichment of Kepi grains with propionibacteria was also evaluated. Apolymerase chain reaction (PCR) based method, specifically designed for therapid identification of propionibacteria, was developed and tested successfully.Using this technique it was concluded that propionibacteria were not a natural partof the Kepi beverage and grains as used in this study. However, during theenrichment of the grains with propionibacteria it was determined that apropionibacteria concentration of 1 x 108 cfu.rnt' was needed for successful PCRamplification results.The data obtained in this study clearly showed that the method by whichKepi is produced, the origin of Kepi grains and the method of Kepi grainpreservation changes the relationship between the microbes constituting thegrains to such an extent that a different microbial community is assembled. It wasalso concluded that traditional methods should be used together with newermethods in determining this microbial community.

[发布机构] Stellenbosch University