Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Thirty lactic acid bacteria were isolated from the intestinal tract of Drosophila simulans Stuvervant and nine lactic acid bacteria from Merlot grapes collected from the same winery in the Stellenbosch region, South Africa. The isolates were grouped according to morphological, biochemical and physiological characteristics. Isolates selected from each group were identified to species level by PCR with species-specific primers, PCR-based DGGE and 16S rDNA sequencing. The majority of isolates from the intestinal tract of Drosophila simulans Stuvervant belonged to the species Lactobacillus plantarum, but Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis and Pediococcus pentosaceus were also identified. As far as we could determine, this is the first report on the isolation of L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis and P. pentosaceus from vinegar flies. Lactobacillus plantarum has previously been isolated from Merlot grapes. The genotypic relatedness among isolates of L. plantarum isolated from the intestinal tract of vinegar flies and from Merlot grapes were determined by RAPD-PCR. The isolates were grouped into four genotypically well-separated clusters. Thirteen isolates from grape must and five from flies yielded identical RAPD-PCR banding patterns and grouped into one cluster, suggesting that they are descendants from the same strain. This suggests that L. plantarum has the ability to use vinegar flies as a vector. / AFRIKAANSE OPSOMMING: Dertig melksuurbakterieë is vanuit die dermkanaal van Drosophila simulans Stuvervant geïsoleer en nege melksuurbakterieë vanuit Merlot-druiwe. Die druiwe is afkomstig van dieselfde wynkelder in die Stellenbosch-area van Suid-Afrika. Die isolate is volgens morfologiese, biochemiese en fisiologiese eienskappe gegroepeer. Verteenwoordigende isolate vanuit die fenotipiese groepe is tot spesievlak met behulp van lukraak ge-amplifiseerde polimorfe-DNA (RAPD) polimerase ketting-reaksie (PKR), PKR met spesie-spesifieke inleiers, PKR-gebaseerde denaturerende gradient-jel elektroforese (DGGE) en 16S rDNA sekwensering geïdentifiseer. Die meerderheid isolate uit die ingewande van Drosophila simulans Stuvervant is as Lactobacillus plantarum geklassifiseer. Stamme van Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis en Pediococcus pentosaceus is ook geïdentifiseer. Sover bekend, is dit die eerste keer dat L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis en P. pentosaceus uit asynvlieë geïsoleer is. Lactobacillus plantarum is voorheen uit Merlot-druiwe geïsoleer. Die genotipiese ooreenkoms tussen die stamme van L. plantarum wat uit die asynvlieë en Merlot-druiwe geïsoleer is, is deur middel van RAPD-PKR bepaal. Hiervolgens is die stamme in vier genotipies goed-gedefinieerde groepe geplaas. Dertien isolate vanuit druiwemos en vyf vanuit asynvlieë het identiese RAPD-PKR bandpatrone vertoon en het in een groep gesorteer. Hierdie resultate dui daarop dat die stamme heel moontlik uit een voorouer ontstaan het en dat asynvlieë heel moontlik as vektor vir L. plantarum dien.

Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations. / AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

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.