PCR-based DGGE typification of the microbial community in Kepi grains

[摘要] ENGLISH ABSTRACT:Kepi is a fermented milk beverage that originated in Eastern Europe. TraditionalKepi is a lightly acidic, carbonated beverage, with a slight yeasty taste. The starterused to produce this beverage is an irregularly shaped, yellowish-white grain-likestructure similar in appearance to a cauliflower floret. The characteristic flavour ofKepi is produced by a complex spectrum of microbial species that include speciesof yeasts, lactic acid bacteria, acetic acid bacteria and mycelial fungi. At the endof the fermentation process the grainy starter can be recovered and re-used, sincethe microbes can easily be recovered as a solid matrix.The microbes comprising Kepi grains have only been identified usingclassical identification techniques such as selective growth media, morphological,physiological and biochemical characteristics. In this study, polymerase chainreaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysiswas used to typify and identify the complex microbial consortium present in theKepi grains. A part of the 168 ribosomal RNA (rRNA) gene from the microbialpopulation in mass-cultured, traditionally cultured and Irish Kepi grains wereamplified using 'Eubacterial' specific primers and a part of the 268 rRNA gene wasamplified using yeast specific primers. The PCR fragments were resolved byDGGE, resulting in unique fingerprints for the Eubacteria and yeasts present in thedifferent Kepi grain types. The traditionally cultured Kepi grains were found toincorporate the most Eubacteria and yeast species, while the mass-cultured Kepigrains contained the lowest number of Eubacteria and yeast species.The different Eubacteria and yeast species were identified by cloning thePCR products and sequencing the cloned inserts. The obtained DNA sequenceswere compared to sequences available on the NCBI website. 8ix lactobacilli wereidentified: Lb. crispatus (KC-4); three Lb. species (KC-36, KC-38 and KC-43); andtwo unculturable lactobacilli (KC-2 and KC-3). The yeasts were identified asSaccharomyces cerevisiae (KC-y18) and Candida lambica (KC-y1). Unidentifiedisolates from kefiran strings that could not be identified using traditional methodswere also identified by cloning the PCR products and sequencing the clonedinserts. The four isolates were identified as Lb. kefiri (KGI-A), Lb. parakefiri (KGIB),Lb. gallina rum (KGI-D) and an unculturable Lactobacillus (KGI-5). The phylogenetic relationship between the identified lactobacilli and thelactobacilli commonly found in Kepi grains was determined. The identifiedlactobacilli were grouped together in a clade with a bootstrap support value of84%. The clade also contained representatives of Lb. delbrueckii subsp. lactis,Lb. acidophilus, Lb. gallinarum, Lb. helveticus, Lb. crispatus, Lb. species andunculturable lactobacilli. The bands in the peR-based DGGE fingerprints of theEubacteria and the yeasts were identified, and a DGGE marker was subsequentlyconstructed for the rapid identification of the Eubacteria present in mass-culturedKepi grains.The data obtained in this study clearly showed that Kepi grains that arecultured differently, as well as Kepi grains from different origins have unique peRbasedDGGE banding patterns for both the Eubacteria and yeasts present in thegrains. The complex microbial consortium comprising Kepi grains could betypified and identified using PeR-based DGGE, DNA cloning and sequencing.The identification of the members of the microbial consortium is of importance forthe future commercialisation of the mass-cultured Kepi grains.

[发布机构] Stellenbosch University