Thesis (MSc Food Sc )--Stellenbsosch University, 2003. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53549 |
| Date | 12 1900 |
| Creators | Garbers, Ilze-Mari |
| Contributors | Witthuhn, R. C., Britz, T. J., Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 75 p. : ill. |
| Rights | Stellenbosch University |
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