Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Kepi is an acidic, self-carbonated milk beverage that is produced by fermenting milk with grain-like structures that contain naturally occurring microbes, including lactic acid bacteria (LAB) and yeasts. The specific microbes present in the Kepi grains are responsible for an acidic-alcoholic fermentation of the milk and also contributes to the various health properties exhibited by Kepi. The combination of microbes in the Kepi grains can vary considerably depending on which type of milk is fermented, the method by which Kepi is produced, the origin of the grains and how the grains are stored. In this study, the impact of various environmental conditions including the different stages during Kepi production, grain origin, Iyophilisation and packaging in three different packaging materials, on the microbial community of Kepi grains were studied using selective growth media, morphology and biochemical characteristics. It was found that there was a general decrease in the microbial counts from laboratory produced Kepi grains, the longer Kepi was produced on a continuous basis. This decrease in microbial counts was also observed during the different stages of Kepi production. The average LAB counts obtained from laboratory produced grains decreased from 1.1 x 108 cfu.q" after 3 d of activation to 6.3 x 107 cfu.q' after 10 d of mass production to 9.7 x 106 cfu.q' after a further 30 d of normal Kepi production. The average yeast counts increased from no detectable yeasts after 3 d of activation to 5.7 x 107 cfu.q' after 10 d of mass production and then decreased again to 7.2 x 106 cfu.q' after 30 d of normal Kepi production. The combination of the isolates varied according to the method by which the Kepi grains were produced and the stress conditions that were applied. Laboratory produced Kepi grains contained the following LAB: Lactobacillus fermentum, 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 Geotrichum candidum. The influence of grain origin on the microbial content of Kepi grains was also investigated using samples of Kepi grains from eight different Southern African sources. The microbial counts of the various Kepi grain samples were found to vary from 6.0 x 105 cfu.q" to 1.7 x 108 cfu.q". Five Lactobacillus, two Leuconostoc, four Candida, one Saccharomyces and a Zygosaccharomyces strain were isolated from these grains, with each grain type having its own unique microbial combination. The microbial content of the Kepi grains that were Iyophilised, packaged in three different packaging materials and stored at room temperature for two months, was very similar. Lactobacillus delbrueckii subsp. delbrueckii was isolated from the Kepi grains packaged in "low density polyethylene film" (LOPE). The grains packaged in "oriented polyester film" (OPET) contained Lb. delbrueckii subsp. delbrueckii and Lb. brevis, while Lb. delbrueckii subsp. delbrueckii and Lb. curvatus was present in the grains packaged in "methallised oriented polyester film" (MOPET). The average microbial counts obtained from the Kepi grains packaged in OPET (2.7 x 106 cfu.q') were only slightly higher than that of the grains packaged in LOPE (1.2 x 106 cfu.q') and OPET (1.4 x 106 cfu.q'). It was concluded that packaging materials for Kepi grains should rather be evaluated on the quality of Kepi produced with the packaged grains than by the specific characteristics of the packaging materials. The enrichment of Kepi grains with propionibacteria was also evaluated. A polymerase chain reaction (PCR) based method, specifically designed for the rapid identification of propionibacteria, was developed and tested successfully. Using this technique it was concluded that propionibacteria were not a natural part of the Kepi beverage and grains as used in this study. However, during the enrichment of the grains with propionibacteria it was determined that a propionibacteria concentration of 1 x 108 cfu.rnt' was needed for successful PCR amplification results. The data obtained in this study clearly showed that the method by which Kepi is produced, the origin of Kepi grains and the method of Kepi grain preservation changes the relationship between the microbes constituting the grains to such an extent that a different microbial community is assembled. It was also concluded that traditional methods should be used together with newer methods in determining this microbial community. / AFRIKAANSE OPSOMMING: Kepi is 'n self-gekarboneerde, effens suur melkdrankie wat geproduseer word deur melk te fermenteer met korrels waarin mikrobes (melksuurbakterieë en giste) natuurlik voorkom. Die mikrobes in die Kepi korrels is verantwoordelik vir 'n suuralkoholiese fermentasie en dra verder by tot die verskeie gesondheidseienskappe wat Kepi besit. Die kombinasie van mikrobes in die Kepi korrels wissel afhangende van die tipe melk wat gebruik word, die metode waarvolgens Kepi gemaak word, die oorsprong van die korrels en hoe die korrels geberg word. In hierdie studie is die impak van verskeie omgewingskondisies insluitende die verskillende stadiums tydens Kepi produksie, korreloorsprong, vriesdroging en verpakking in drie verskillende verpakkingsmateriale, op die mikrobiese samestelling van Kepi korrels bepaal m.b.v. selektiewe groei media en morfologiese en biochemiese eienskappe. Dit is gevind dat daar 'n afname was in die mikrobiese tellings van laboratorium geproduseerde Kepi korrels hoe langer Kepi op 'n aaneenlopende basis geproduseer is. Die afname in mikrobiese tellings is ook waargeneem tydens die verskillende stadiums van Kepi produksie. Die gemiddelde melksuurbakterieë tellings van laboratorium geproduseerde korrels het afgeneem vanaf 1.1 x 108 kve.q' na 3 d van aktivering tot 6.3 x 107 kve.q" na 10 d van massakweking tot 9.7 x 106 kve.q" na 'n verdere 30 d van normale Kepi produksie. Die gemiddelde gis tellings het gestyg vanaf geen giste na 3 d van aktivering tot 5.7 x 107 kve.q" na 10 d van massakweking en het toe weer gedaal tot 7.2 x 106 kve.q' na 30 d van normale Kepi produksie. Die kombinasie van die isolate het gewissel na gelang van die metode waarop die Kepi korrels geproduseer is en die stres kondisies wat toegepas is. Laboratorium geproduseerde Kepi korrels het bestaan uit Lactobacillus fermentum, Lb. brevis 3, Lb. p/antarum, Lb. de/brueckii subsp. de/brueckii, Lactococcus /actis subsp. /actis 1en Leuconostoc mesenteroides subsp. cremoris. Die giste en misiliëre fungi wat geïs~leer is was 'n Zygosaccharomyces stam, Cryptococcus humico/us, Candida lambica, C. krusei, C. kefyr en Geotrichum candidum. Die invloed wat die oorsprong van Kepi korrels op die mikrobiese samestelling daarvan het, is bepaal m.b.v. Kepi korrels afkomstig van agt verskillende dele in Suidelike Afrika. Die mikrobiese tellings van die verskeie tipes Kepi korrels het gewissel vanaf 6.0 x 105 kve.q' tot 1.7 x 108 kve.q", Vyf Lactobacillus, twee Leuconostoc, vier Candida, een Saccharomyces en 'n Zygosaccharomyces is geïsoleer vanuit die korrels, waarvan elke tipe korrel sy eie unieke mikrobiese samestelling gehad het. Die mikrobiese samestelling van korrels wat gevriesdroog, verpak is in drie verskillende verpakkingsmateriale en by kamertemperatuur gestoor is vir twee maande, was baie eenders. Vanuit die Kepi korrels wat verpak is in "lae digtheid polietileen film" (LOPE) is Lb. delbrueckii subsp. teetis geïsoleer. Die korrels wat verpak is in "georienteerde poltester film" (OPET) het Lb. delbrueckii subsp. leetis en Lb. brevis besit, terwyl Lb. delbrueckii subsp. leetis en Lb. curvatus teenwoordig was in die korrels wat in "gemetileerde georienteerde poltester film" (MOPET) verpak is. Die gemiddelde mikrobiese tellings van die korrels wat verpak is in OPET (2.6 x 106 kve.q') was effens hoër as dié van die korrels wat verpak is in LOPE (1.2 x 106 kve.q") en MOPET (1.3 x 106 kve.q"). Dit is bepaal dat verpakkingsmateriale vir Kepi korrels eerder geevalueer moet word op die kwaliteit van die Kepi wat met die verpakte korrels geproduseer word, as op die spesifieke eienskappe van die verpakkingsmateriale. Die mikrobiese verryking van Kepi korrels met propionibakterieë is ook ondersoek. 'n Polimerase ketting reaksie (PKR) gebaseerde metode, spesifiek ontwerp vir die vinnige identifikasie van propionibakterieë, is ontwikkel en suksesvol getoets. Met hierdie tegniek is bepaal dat propionibakterieë nie 'n natuurlike deel is van die Kepi drankie en korrels soos gebruik in hierdie studie. Gedurende die verryking van Kepi korrels met propionibakterieë is dit egter ook bepaal dat 'n propionibakterieë konsentrasie van 1 x 108 kve.rnl' nodig is vir suksesvolle PKR amplifikasie resultate. Die data verkry in hierdie studie het duidelik gewys dat die metode van Kepi produksie, die oorsprong van Kepi korrels en die metode waarop Kepi korrels gepreserveer word, verander die verhouding tussen die mikrobes in die korrels tot so 'n mate dat 'n nuwe mikrobiese gemeenskap saamgestel word. Die gevolgtrekking is ook gemaak dat tradisionele metodes saam met nuwer metodes gebruik moet word in die bepaling van hierdie mikrobiese gemeenskap.

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.

Thesis (MSc Food Sc )--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Kepi is a refreshing, fermented dairy beverage that differs from other fermented milk products in that it is produced with a mixed microbial community which is confined to discrete grains. These grains can be recovered as a solid matrix at the end of the fermentation and then be reutilised as a starter to ferment the next batch of milk. The grain microbial community consists of a symbiotic association of yeasts and lactic acid bacteria, but the overall composition of the grains has not been completely elucidated. The microbes in the grains are embedded in a protein-polysaccharide Kefiran matrix, which appears essential for grain formation. The mechanism of grain formation is still not fully understood and it thus remains undecided which organism is really responsible for the production of this proteinpolysaccharide matrix. The aim of this study was to isolate, characterise and identify the microbes present in Kefiran from mass cultured South African grains and then to evaluate grain formation with these purified cultures isolated from Kefiran strings using a mass cultivation process. Sixteen strains of lactic acid bacteria and one yeast strain were isolated from Kefiran strings produced during the mass cultivation of South African Kepi grains. API technology, numerical clustering and DNA sequence comparisons were used to identify the purified isolates. The isolates were grouped into seven clusters by numerical clustering and clustering distance from selected reference and marker strains. The heterofermentative lactobacilli were identified as Lactobacillus parakefiri and Lb. kefiri and the homofermentative strains as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus and Lb. bavaricus. One isolate was found to be a member of the genus Lactobacillus, but was not positively identified to species level. Cultures isolated from Kefiran were evaluated for ability to grain formation by adding 1 x 109 cfu.ml:' bacteria and 1 x 108 cfu.ml' yeast to double pasteurised, full cream milk during the mass cultivation process. It was found that the control and all the cultures in double pasteurised milk showed grain accumulation indicating that other microbes were present in pasteurised and double pasteurised milk which had an influence on the grain forming ability. The cultures isolated from pasteurised and double pasteurised milk included members of the species Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliermondii, Chryseobacterium meningosepticum, Pseudomonas putida and four isolates of the Bacillus cereus group. It was found that these rod-shaped "milk isolates" resulted in grain accumulation when inoculated into UHT milk and it was concluded that the "milk isolates" did contribute to grain formation. These isolates were then combined with the Kefiran cultures and this resulted in grains very similar to the traditional Kepi grains. These grains were made from Lb. gallinarum in double pasteurised milk as well with a combination of Lb. gallinarum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica and Pseudomonas putida in URT milk. The grains were firm, elastic and did not dissolve in water but kept their structure and were retained when sieved. An acceptable Kepi beverage was produced from these grains. From these typically traditional grain characteristics it was concluded that, even though the microbial compositions were probably not the same, the general appearance was similar to traditional grains and that it is thus possible to produce grains from pure single strain Kefiran cultures and "milk isolates". Furthermore, it was possible to produce a Kepilike beverage from these grains, which included similar characteristics as the traditional Kepi beverage. / AFRIKAANSE OPSOMMING: Kepi is "n verfrissende, gefermenteerde suiweldrankie wat van ander gefermenteerde produkte verskil in die opsig dat dit vervaardig word deur Kepi korrels in melk te inkubeer. Die Kepi korrels kan aan die einde van die fermentasie herwin word en weer gebruik word om die volgende lot melk te fermenteer. Die korrels bestaan uit "n simbiotiese samestelling van giste en melksuurbakterieë, maar die presiese samestelling van die korrels is steeds onbekend. Die mikro-organismes is vasgevang in "n proteïen-polisakkaried Kefiran matriks en die Kefiran word as essensieel beskou vir korrelvorming. Die meganisme van korrelvorming bly steeds onbekend en daar is nog nie tot "n gevolgtrekking gekom oor watter organisme die Kefiran produseerder is nie. Die doel van die studie was om die mikro-organismes in Kefiran te isoleer en te identifiseer deur Suid-Afrikaanse Kepi korrels te massa kweek. Hierdie mikroorganismes was dan verder geëvalueer ten opsigte van korrel vorming. Sestien melksuurbakterieë isolate en een gis isolaat is geïsoleer vanuit die Kefiran. API tegnologie, numeriese groepering en DNA volgorde vergelykings was gebruik om die isolate te identifiseer. Die isolate is in sewe groepe verdeel volgens numeriese groepering. Die afstand van verwysings en merker organismes is ook in ag geneem. Die heterofermentatiewe organismes is geïdentifiseer as Lactobacillus parakefiri en Lb. kefiri en die heterofermentatiewe organismes as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus en Lb. bavaricus. Een isolaat kon nie geïdentifiseer word tot op spesie vlak nie, maar is verwant aan die genus Lactobacillus. Hierdie geïsoleerde Kefiran kulture is geëvalueer ten op sigte van korrelvorming, deur 1 x 109 kve.ml' van die bakterieë en 1 x 108 kve.ml' van die gis by dubbel gepasteuriseerde volroom melk te voeg tydens die massakwekings proses. Die kontrole wat geen bygevoegde kulture bevat nie, sowel as die wat wel bygevoegde kulture bevat, het korrel vorming getoon. Laasgenoemde toon dat daar organismes teenwoordig is in gepasteuriseerde en dubbel gepasteuriseerde melk wat "n rol kan speel tydens korrelvorming. Die kulture wat geïsoleer is vanuit gepasteuriseerde en dubbel gepasteuriseerde melk, sluit in: Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliennondii, Chryseobacterium menigosepticum, Pseudomonas putida en vier isolate van die Bacillus cereus groep. Hierdie organismes wat uit melk geïsoleer is, het korrelvorming getoon in UHT melk en die gevolgtrekking kan gemaak word dat die "melk organismes" wel "n rol speel tydens korrel vorming. Hierdie "melk isolate" in kombinasie met die Kefiran kulture het korrels tot gevolg gehad wat baie dieselfde was as tradisionele Kepi korrels. Laasgenoemde korrels is gemaak deur Lb. gallina rum in dubbel gepasteuriseerde melk, sowel as deur "n kombinasie van Lb. gallina rum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica en Pseudomonas putida in UHT melk. Die korrels was stewig, elasties, het nie opgelos in water nie en het hulle struktuur behou wanneer gesif. Wanneer hierdie tipiese tradisionele korrels se eienskappe in ag geneem word, kan die gevolgtrekking gemaak word dat alhoewel die mikrobiese samestelling van die korrels nie dieselfde is as die tradisionele korrel nie, is die algemene voorkoms en eienskappe dieselfde en dat dit wel moontlik is om korrels te produseer deur isolate geïsoleer vanuit Kefiran en melk. Verder was dit moontlik om "n drankie te vervaardig met die korrels wat baie dieselfde is as tradisionele Kepi.