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.