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

Garbers, Ilze-Mari (2003-12)

Thesis (MSc Food Sc )--Stellenbsosch University, 2003.

Thesis

ENGLISH ABSTRACT: Kepi is a fermented milk beverage that originated in Eastern Europe. Traditional Kepi is a lightly acidic, carbonated beverage, with a slight yeasty taste. The starter used to produce this beverage is an irregularly shaped, yellowish-white grain-like structure similar in appearance to a cauliflower floret. The characteristic flavour of Kepi is produced by a complex spectrum of microbial species that include species of yeasts, lactic acid bacteria, acetic acid bacteria and mycelial fungi. At the end of the fermentation process the grainy starter can be recovered and re-used, since the microbes can easily be recovered as a solid matrix. The microbes comprising Kepi grains have only been identified using classical identification techniques such as selective growth media, morphological, physiological and biochemical characteristics. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis was used to typify and identify the complex microbial consortium present in the Kepi grains. A part of the 168 ribosomal RNA (rRNA) gene from the microbial population in mass-cultured, traditionally cultured and Irish Kepi grains were amplified using 'Eubacterial' specific primers and a part of the 268 rRNA gene was amplified using yeast specific primers. The PCR fragments were resolved by DGGE, resulting in unique fingerprints for the Eubacteria and yeasts present in the different Kepi grain types. The traditionally cultured Kepi grains were found to incorporate the most Eubacteria and yeast species, while the mass-cultured Kepi grains contained the lowest number of Eubacteria and yeast species. The different Eubacteria and yeast species were identified by cloning the PCR products and sequencing the cloned inserts. The obtained DNA sequences were compared to sequences available on the NCBI website. 8ix lactobacilli were identified: Lb. crispatus (KC-4); three Lb. species (KC-36, KC-38 and KC-43); and two unculturable lactobacilli (KC-2 and KC-3). The yeasts were identified as Saccharomyces cerevisiae (KC-y18) and Candida lambica (KC-y1). Unidentified isolates from kefiran strings that could not be identified using traditional methods were also identified by cloning the PCR products and sequencing the cloned inserts. 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 the lactobacilli commonly found in Kepi grains was determined. The identified lactobacilli were grouped together in a clade with a bootstrap support value of 84%. The clade also contained representatives of Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallinarum, Lb. helveticus, Lb. crispatus, Lb. species and unculturable lactobacilli. The bands in the peR-based DGGE fingerprints of the Eubacteria and the yeasts were identified, and a DGGE marker was subsequently constructed for the rapid identification of the Eubacteria present in mass-cultured Kepi grains. The data obtained in this study clearly showed that Kepi grains that are cultured differently, as well as Kepi grains from different origins have unique peRbased DGGE banding patterns for both the Eubacteria and yeasts present in the grains. The complex microbial consortium comprising Kepi grains could be typified and identified using PeR-based DGGE, DNA cloning and sequencing. The identification of the members of the microbial consortium is of importance for the future commercialisation of the mass-cultured Kepi grains.

AFRIKAANSE OPSOMMING: Kepi is 'n gefermenteerde melkdrankie wat sy oorsprong het in Oos Europa. Tradisionele Kepi is 'n effens suur, gekarboneerde drankie wat effens na gis smaak. Die beginkultuur wat gebruik word om dié drankie te maak is 'n oneweredige, geel-wit korrelagtige struktuur wat baie lyk soos 'n blomkoolkoppie. Die karakteristieke smaak van Kepi word geproduseer deur 'n komplekse spektrum mikrobiese spesies wat giste, melksuur- en asynsuurbakterieë en ~. misillêre fungi insluit. Aan die einde van die fermentasieproses kan die korrelagtige beginkultuur herwin word en weer gebruik word, aangesien die mikrobes maklik herwin kan word as 'n soliede matriks. Die mikrobes waaruit Kepikorrels bestaan, is nog slegs met behulp van klassieke identifikasiemetodes soos selektiewe groeimedia, morfologiese, fisiologiese and biochemiese eienskappe geïdentifiseer. In hierdie studie is polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise gebruik om die komplekse mikrobiologiese konsortium in die Kepikorrels te tipeer en te identifiseer. 'n Gedeelte van die 16S ribosomale RNS (rRNS) geen van die mikrobiologiese populasie in massagekweekte, tradisioneel gekweekte en Ierse Kepikorrels is geamplifiseer met 'Eubakferiële' spesifieke peilers en In gedeelte van die 26S rRNS geen is geamplifiseer met gis spesifieke peilers. Die PKR fragmente is onderskei deur DGGE, wat unieke vingerafdrukke vir die Eubakteriële- en gisspesies in die verskillende Kepikorrel tipes gelewer het. Die tradisioneel gekweekte Kepikorrels het die meeste Eubakteriële- en gisspesies geïnkorporeer, terwyl die Ierse Kepikorrels die minste Eubakteriële- en gisspesies geïnkorporeer het. Die verskillende Eubakteriële- en gisspesies is geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgordes te bepaal. Die ONS volgordes is dan vergelyk met volgordes wat op die NCSI webwerf beskikbaar is. Ses lactobacilli is geïdentifiseer: Lb. ctispetus (KC-4); drie Lb. spesies (KC-36, KC-38 en KC-43); en twee onkultiveerbare lactobacilli (KC-2 en KC-3). Die giste is geïdentifiseer as Saccharomyces cerevisiae (KC-y18) en Candida lambica (KC-y1). Ongeïdentifiseerde isolate van kefiranstringe is ook geïdentifiseer deur klonering van die PKR produkte en deur die gekloneerde insetsels se volgorde te bepaal. Dié vier isolate is geïdentifiseer as Lb. kefiri (KGIA), Lb. parakefiri (KGI-B), Lb. gallina rum (KGI-D) en 'n onkultiveerbare Lactobacillus (KGI-5). Die filogenetiese verwantskap is bepaal tussen die geïdentifiseerde lactobacilli en lactobacilli wat geredelik in Kepikorrels gevind word. Die geïdentifiseerde lactobacilli was saam in 'n groep gegroepeer met 'n bootstrap waarde van 84%. Die groep het ook verteenwoordigers van Lb. delbrueckii subsp. lactis, Lb. acidophilus, Lb. gallina rum, Lb. helveticus, Lb. crispatus, Lb. species en 'n onkultiveerbare laktobacilli ingesluit. Die bande in die PKR-gebaseerde DGGE vingerafdrukke van die Eubakterieë en die giste is geïdentifiseer, en 'n DGGE merker is gemaak vir die vinnige identifikasie van die Eubakterieë wat in die massagekweekte Kepikorrels teenwoordig is. Die data wat in die studie verkry, is wys duidelik dat Kepikorrels wat op verskillende maniere gekweek is, en wat verskillende oorspronge het, unieke PKR-gebaseerde DGGE bandpatrone het vir beide die Eubakterieë en giste wat in die korrels teenwoordig is. Die komplekse mikrobiologiese konsortium waaruit Kepikorrels bestaan kon getipeer en geïdentifiseer word deur PKR-gebaseerde DGGE, klonering van DNS en volgordebepaling. Die identifikasie van lede van die mikrobiologiese konsortium is belangrik vir die toekomstige kommersialisasie van die massagekweekte Kepikorrels.

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