Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in the production of a variety of fermented products. Many of these bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically closely related to the producer cell. Since many of these target bacteria include foodbome pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the practical importance of these peptides as food preservatives has been well documented and, in the case of nisin and pediocin PA-I, commercially explored. The increased demand from health conscious consumers for foods with no chemical preservatives is putting renewed pressure on the producer to supply a "clean and green" product, but with the same or even an extended shelf life. Various research groups are screening lactic acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds. Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence in the N-terminal region. Little is known about the production and mode of activity of pediocin PD-I. In this study, production of pediocin PD-I was significantly increased by optimizing the growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone (1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH during fermentation stimulated pediocin PD-I production and the level of organic acids produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the latter medium. Under these conditions the specific bacteriocin activity increased by a factor of approximately six after 55 h of fermentation. The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin removed the biofilms from the surfaces and reduced the biomass either completely, as in the case of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively. These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified Chardonnay must medium. To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being very effective in controlling the cell numbers of free-living cells of 0. oeni, the three bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the production and activity levels ofpediocin PD-I could be increased without genetically modifying the producer strain is an added advantage. / AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer. Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik. Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer, maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten einde Gram-negatiewe bakterieë, giste en swamme te inhibeer. Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met 'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor die meganisme van werking van hierdie bakteriosiene. In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween 80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die bakteriosien met 'n faktor van ongeveer ses verhoog. Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum 423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50% van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000 AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem. Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the quality and wholesomeness of the final product. Yeasts are primarily responsible for alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is conducted by lactic acid bacteria. These bacteria are important in winemaking and can have a positive or negative effect on the wine quality. Biogenic amines are one of the compounds produced by lactic acid bacteria, which affect the hygienic quality and wholesomeness of the wine negatively and directly pose a health risk to the consumer. The demand of consumers for higher quality and healthier foods has led to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide variety of food products, such as cheese, dried sausage, sauerkraut, fishery products, chocolates, wine and beer. This thesis focussed on the presence of biogenic amines in wine. The first objective of the study was to determine the ability of lactic acid bacteria isolated from South African wine to produce biogenic amines, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. The results obtained showed that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be the lactic acid bacteria responsible for tyramine and putrescine production and that it can contribute significantly to the overall biogenic amine content in wines. The results also suggest that amine production is strain dependent and not species specific. None of the lactic acid bacteria tested had the ability to produce histamine or cadaverine. It is important to remember that the ability of the lactic acid bacteria to produce biogenic amines has only been investigated in synthetic media and that it does not necessarily imply similar behaviour in wine. Wine represents a complex environment with a wide number of factors influencing microbial growth and decarboxylase activity and, thus, further investigation is necessary to determine if these amine-producing bacteria behave similarly in wine conditions. In addition, the polymerase chain reaction (PCR) amplification method was used for the identification of the tyrosine decarboxylase (TOe) gene in some of the tyramine-producing lactic acid bacteria. This was followed by the sequencing of the amplified products, which are partial TOe gene sequences, of two L. brevis strains and of a L. hilgardii strain. Only one tdc gene sequence has been described for bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis lOEB 9809 was described. An amino acid sequence alignment of the three TOe gene fragments, obtained in this study, with the known TOe gene fragment of L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of relatedness and conserved regions. To meet consumer demands, procedures are necessary to prevent the formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species involved in the winemaking process. Another possible way would be to develop a rapid detection method for bacteria carrying amino acid decarboxylase genes. The results of this study provide knowledge about which lactic acid bacteria in the winemaking process could contribute to the production of biogenic amines and the sequencing of additional partial TOe genes could possibly assist in the development of a rapid detection method for tyramine-producing lactic acid bacteria in food products. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê. Biogeniese amiene is een van die komponente wat deur melksuurbakterieë geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade, wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in wyn. Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid- Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde gedrag in wyn sal toon. Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809 en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van amiene in voedselprodukte voorkom word. Een manier van voorkoming is om amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte bydra.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Lactobacillus plantarum 285, isolated from sorghum beer, produces bacteriocin 285, which displays activity against several food spoilage organisms. For future application of bacteriocin 285 in the food industry, it was important to characterize the peptide and identify the genes encoding its production. The effect of bacteriocin 285 on sensitive cells was determined through the use of an indicator (sensitive) organism, Lactobacillus sakei DSM 20017. The indicator strain was genetically modified to express GFP (green fluorescent protein), with the aim of quantifying the antibacterial activity of bacteriocin 285 as a function of GFP fluorescence. Bacteriocin 285 proved to be identical to plantaricin 423 produced by L. plantarum 423. Plantaricin 423 is a class lIa bacteriocin and displays antimicrobial activity towards a broad spectrum of bacteria, including several food spoilage organisms. The sensitivity of L. sakei DSM 20017 towards antibacterial peptides produced by Lactobacillus curvatus DF38, L. plantarum 285, Lactobacillus casei LHS and Lactobacillus salivarius 241 is not limited to the growth stage of the organism. Cells remained sensitive to all four of these bacteriocins, from lag phase to late exponential growth. To inhibit growth of up to 90% of the cells of L. sakei DSM 20017, 1 AU/ml bacteriocin 285 (7 ng/ml) of partially purified bacteriocin 285 was required. However, to kill all viable cells of L. sakei DSM 20017, 16 AU/ml (110 ng/ml) of partially purified bacteriocin 285 was required. The gfpuv gene, encoding GFPuv, was cloned downstream of the Idh promoter and successfully expressed in L. sakei DSM 20017. However, GFPuv fluorescence could not be used as a direct method to quantify the antimicrobial activity of bacteriocin 285, since cells of strain DSM 20017 remained fluorescent for prolonged periods after treatment with lethal concentrations of the bacteriocin. The non-viability of the cells was confirmed with epifluorescence microscopy and a L1VE/DEAD® Baclight™ Bacterial Viability Probe. Cells that were stained with the viability probe indicated that the majority of untreated L. sakei DSM 20017 cells were viable. However, treatment of strain DSM 20017 with 16 AU/ml bacteriocin 285 rendered all visible cells non-viable. / AFRIKAANSE OPSOMMING: Lactobacillus plantarum 285 wat uit sorgumbier geïsoleer is, produseer bakteriosien 285. Die bakteriosien toon aktiwiteit teen verskeie organismes wat voedselbederi veroorsaak. Vir toekomstige aanwending van bakteriosien 285 in die voedselindustrie was dit belangrik om die peptied te karakteriseer en die gene wat vir die produksie daarvan kodeer, te identifiseer. Die effek van bakteriosien 285 op sensitiewe selle is bepaal deur die gebruik van 'n indikator (sensitiewe)-organisme, Lactobacillus sakei DSM 20017. Die indikator-organisme is geneties verander om die GFP (groen fluoreserende proteïen) uit te druk. Die doel was om die antibakteriese aktiwiteit van bakteriosien 285 te kwantifiseer as 'n funksie van GFP fluorisensie. Bakteriosien 285 is identies aan plantarisien 423 wat deur L. plantarum 423 produseer word. Plantarisien 423 is 'n klas Iia bakteriosien en vertoon antimikrobiese aktiwiteit teenoor 'n wye verskeidenheid bakterieë, insluitende verskeie organismes wat voedsel bederf. Die sensitiwiteit van L. sakei DSM 20017 teenoor antibakteriese peptiede wat deur Lactobacillus cutveius DF38, L. plantarum 285, Lactobacillus casei LHS en Lactobacillus salivarius 241 geproduseer word, word nie beïnvloed deur die groeifase van die organisme nie. Selle het sensitief gebly teenoor al vier die bakteriosiene van sloer- tot laat eksponensiële groei. Om groei van tot 90% van L. sakei DSM 20017 selle te inhibeer, word 1 AU/ml (7 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Om alle lewensvatbare L. sakei DSM 20017 selle te dood, word 16 AU/ml (110 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Die gfpuv-geen, wat GFPuv kodeer is stroomaf van die Idh-promoter gekloneer en suksesvol in L. sakei DSM 20017 uitgedruk. GFPuv fluoresensie kon nie as direkte metode gebruik word om die antimikrobiese aktiwiteit van bakteriosien 285 te bepaal nie, aangesien die selle van L. sakei DSM 20017 fluoreserend gebly het lank na behandeling met dodelike konsentrasies van die bakteriosien. Die lewensvatbaarheid van die selle is bevestig deur epifluoresensiemikroskopie en 'n LlVE/DEAD® Bac/ight™ bakteriese lewensvatbaarheidspeiler. Selle van L. sakei DSM 20017 wat deur die peiler gekleur is, het gewys dat die meeste selle wat nie deur bakteriosien 285 behandel was nie, lewensvatbaar was. Behandeling van L. sakei DSM 20017 met 16 AU/ml bakteriosien 285 het al die sigbare selle gedood.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Barley malt is the predominant raw material for beer brewing world-wide. To meet consumer demand, a constant high quality malt product is required. Malt quality is determined by the degree of substrate hydrolysis during germination and mashing which serves as fermentable substrates for alcoholic fermentation during brewing. It is often difficult to sustain malt of high quality due to inconsistent malt batches and poor germination capacities of dormant barley. External additives such as chemicals and gibberellic acid have been used to overcome these difficulties but are unwanted in the beverage industry. Maltsters are consequently always in search of alternative solutions. Microbes produce diverse enzymes which can contribute to substrate hydrolysis during germination. The development of such starter cultures might provide a natural and economically feasible alternative to augment barley germination. Starter culture technology has been employed in the malting industry, although the main focus has been to improve the microbial stability of malt. The exploitation of cultures with hydrolytic capabilities to augment barley germination is consequently largely unexplored. The aim of this study was to develop a starter culture which can contribute to the enzymatic degradation of barley polymers. Geotrichum spp. and Lactobacillus plantarum were isolated from substrates rich in polymers present in barley and screened for enzymatic capabilities. Geotrichum spp. produced cellulase, xylanase, protease and β-glucanase activities, while L. plantarum harboured cell-bound and extracellular α-amylase activities. These cultures were added in different combinations during the malting of Erica and SSG 564 cultivars, but did not enhance germination significantly. Improved malt parameters did not correlate with microbial enzyme activities and the data were not repeatable. Preliminary plate assays could thus not be used to predict enzyme production in a malting environment. Cell-free supernatants with known enzyme activities of Aspergillus sp., Trichoderma reesei and Rhizopus sp. significantly enhanced malt quality. To our knowledge, the use of fungal supernatant to augment malt modification is a novel concept. Supernatant is more convenient than starter cultures and will aid to deliver more constant malt products than live cultures, as known enzyme levels are added. / AFRIKAANSE OPSOMMING: Garsmout is wêreldwyd die oorheersende roumateriaal vir bier brou. Om die aanvraag van verbruikers te bevredig, word 'n konstante hoë gehalte mout produk vereis. Die kwailiteit van mout word bepaal deur die graad van substraathidrolise gedurende ontkieming, wat dien as fermenteerbare substraat vir alkoholiese fermentasie tydens verbrouing. Dit is dikwels moeilik om ʼn konstante, hoë gehalte, moutproduk te lewer as gevolg van variasie in mout en die swak ontkiemingsvermoë van dormante gars. Hierdie probleem kan oorbrug word met eksterne toevoegings soos chemikalieë en gibberelliensuur, maar dit is nie ʼn gewensde praktyk in die broubedryf nie. Vermouters is gevolglik gedurig op soek na alternatiewe oplossings. Mikroörganismes produseer diverse ensieme wat kan bydra tot substraathidrolise gedurende ontkieming. Die ontwikkeling van sodanige suurselkulture is moontlik 'n natuurlike en ekonomies praktiese alternatief om die ontkieming van gars te stimuleer. Suurselkulture is reeds in die moutindustrie gebruik, alhoewel die fokus hoofsaaklik was om die mikrobiese stabiliteit van mout te verbeter. Die konsep om kulture met hidrolitiese vermoëns te gebruik om garsontkieming aan te vul is gevolglik grootliks onverken. Die doel van hierdie studie was om 'n suurselkultuur te ontwikkel wat kan bydra tot 'n ensiematiese afbraak van die polimere in gars. Geotrichum spp. en Lactobacillus plantarum is uit substrate ryk aan polimere teenwoordig in gars geïsoleer en vir hul ensiem aktiwiteite getoets. Geotrichum spp. het sellulase, xylanase, protease en β-glukanase aktiwiteit getoon, terwyl L. plantarum sel-gebonde en ekstrasellulêre α-amilase aktiwiteit getoon het. Hierdie kulture is in verskillende kombinasies tydens die vermouting van Erica en SSG 564 kultivars bygevoeg, maar het nie tot ʼn verbetering in die ontkieming van die gars gelei nie. Geen korrelasie is gevind tussen verbeterde mout parameters en mikrobiese ensiemaktiwiteit nie. Die resultate was ook nie herhaalbaar nie. Voorlopige plaattoetse kan dus nie as 'n maatstaf gebruik word om ensiem produksie deur suurselkulture in vermounting te voorspel nie. Sel-vrye supernatante van Aspergillus sp., Trichoderma reesei en Rhizopus sp., met bekende ensiem aktiwiteit, het die gehalte van mout aansienlik verbeter. Sover ons kennis strek is die gebruik van supernatante van fungi om die ontkieming van gars te stimuleer ʼn nuwe konsep. Supernatant is meer gerieflik as suurselkulture en sal help om konstante mout produkte te lewer aangesien ensiemvlakke beter beheer kan word.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Wine quality is compromised by the presence of 3-hydroxypropionaldehyde (3-HPA) due to spontaneous conversion into acrolein under wine making conditions. Acrolein is highly toxic and is presence has been correlated with the development of bitterness in wine. Lactic acid bacterial strains isolated from South African red wine, Lactobacillus pentosus and Lactobacillus brevis, are implicated in accumulating 3- HPA during anaerobic glycerol fermentation. The environmental conditions leading to its accumulation are elucidated. In aqueous solution 3-HPA undergoes reversible dimerization and hydration, resulting in an equilibrium state between different derivatives. Interconversion between 3-HPA derivatives and acrolein is a complex and highly dynamic process driven by hydration and dehydration reactions. Acrolein is furthermore highly reactive and its steady-state concentration in complex systems very low. As a result analytical detection and quantification in solution is problematic. This study highlights the roles played by natural chemical derivatives and shows that the acrolein dimer can be used as a marker for indicating the presence of acrolein in wines. Solid-phase microextraction (SPME) coupled to gas chromatograph mass spectrometry (GC-MS) was validated as a technique for direct detection of the acrolein dimer in wine. The potential of a recently introduced sorptive extractive technique with a sample enrichment probe (SEP) was also investigated. The SPME technique simplifies the detection process and allows for rapid sampling of the acrolein marker, while SEP is more sensitive. / AFRIKAANSE OPSOMMING: Die teenwoodigheid van 3-hidroksiepropioonaldehied (3-HPA) in wyn het ‘n negatiewe invloed op kwaliteit as gevolg van die moontlike omskakeling na akroleien tydens die wynmaak prosses. Akroleien is hoog toksies en is moontlik betrokke by die ontwikkeling van ‘n bitter komponent in wyn. Hierdie studie wys dat stamme van die melksuurbakteriëe Lactobacillus pentosus en Lactobacillus brevis, geisoleer uit Suid-Afrikaanse wyn, 3-HPA tydens anaerobiese alkoholiese fermentasie kan opbou. Kondisies wat ontwikkeling beinvloed is bestudeer. 3-HPA ondergaan omkeerbare dimerisasie en hidrasie in oplossing en het ‘n ewewig tussen veskillende derivate tot gevolg. Omkakeling tussen 3-HPA derivate en akroleien is ‘n komplekse en hoogs dinamiese prosses wat gedryf word deur hidrasie en dehidrasie reaksies. Akroleien is verder hoogs reaktief en die ewewigskonsentrasie van hierdie aldehied in komplekse omgewings is laag. Analitiese waarneming en kwantifisering is gevolglik problematies. Hierdie studie lig die rol wat natuurlike chemise derivate speel duidelik uit en wys dat die akroleien dimeer as ‘n merker gebruik kan word om die teenwoodigeid van akoleien in wyn te staaf. Soliede-fase mikro-ekstraksie (SPME) gekoppel aan gas chromatografie massa spektroskopie (GC-MS) is gevalideer as ‘n tegniek vir die direkte waarneming van die akroleien dimeer in wyn. Die potensiaal van ‘n nuwe ekstraksie tegniek, gebasseer op ‘n peiler wat vir die monster verreik (SEP), was ook ondersoek. Die SPME tegniek is vinnig en vergemaklik analiese, terwyl SEP meer sensitief is.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Spoilage, be it due to microbial actions, chemical reactions or both, poses a serious threat to the food and beverage industries. Not only can spoilage lead to great economic losses, but it can also cause industries to lose their competitive edge in the economic and consumer market. Considering all the modern technologies and the range of preservation techniques that are available, it is surprising that spoilage is still an economic problem. Wine spoilage due to unpalatable bitterness, and the role of lactic acid bacteria (LAB) in causing this bitterness, have received much attention over the years, but no definite understanding has yet emerged. The first objective of this study was to isolate, enumerate and identify the LAB from three red grape varieties, viz. Pinotage, Merlot and Cabernet Sauvignon. The LAB populations on the grapes of all three varieties ranged from 102 to 104 cfu/ml during the 2001 and 2002 harvest seasons. The Cabernet Sauvignon grapes had slightly higher numbers than the Pinotage and Merlot. The LAB population in the Cabernet Sauvignon, Pinotage and Merlot wines after completion of the alcoholic fermentation ranged from 102 to 105 cfu/ml, while during 2002 the numbers in wine undergoing malolactic fermentation (MLF) ranged from 104 to 108 cfu/ml. The isolated LAB were divided into the three metabolic groups, with 59% belonging to the facultatively heterofermentative group, 26% to the obligately heterofermentative group and 15% to the obligately homofermentative group. The isolates were identified by means of species-specific primers as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lb. hilgardii (15), Lb. plantarum (98), Lb. pentosus (12), Lb. paraplantarum (3), Lb. paracasei (28), Pediococcus acidilactici (2) and Pediococcus spp. (35). The most predominant species isolated was Lb. plantarum, followed by Pediococcus spp. The results suggest that Pinotage carries a more diverse LAB population in comparison to Merlot and Cabernet Sauvignon. The second objective of this study was to determine the presence of the glycerol dehydratase gene in the LAB strains by using the G01 and G02 primers. Twenty-six strains tested positive, namely Lb. plantarum (15), Lb. pentosus (1), Lb. hilgardii (5), Lb. paracasei (2), Lb. brevis (2) and a Pediococcus spp. (1). Interestingly, 62% of these strains were isolated from Pinotage. The strains all had the ability to degrade glycerol by more than 90%, and no significant differences were observed between the species. The GO-possessing strains exhibited varying degrees of inhibition towards Gram-positive and Gram-negative bacteria, and the results suggest that this inhibition activity may be similar to that of reuterin, which is produced by Lb. reuteri. This study can form the foundation for unravelling the causes of bitterness in red wines. Combining the results of this study with analytical, sensory and molecular data may very well provide the industry with valuable tools with which to combat the occurrence of bitterness. / AFRIKAANSE OPSOMMING: Bederf as gevolg van mikrobiese aksies, chemiese reaksies of beide, hou 'n groot bedreiging vir die voedsel- en drankbedrywe in. Nie net kan bederf lei tot groot ekonomiese verliese nie, maar dit kan ook veroorsaak dat bedrywe hul kompeterende voordeel in die ekonomiese en verbruikersmarkte verloor. As die moderne tegnologie en die reeks preserveringstegnieke wat beskikbaar is, in ag geneem word, is dit verbasend dat bederf steeds 'n ekonomiese probleem is. Wynbederf as gevolg van oormatige bitterheid en die rol van melksuurbakterieë (MSB) in die ontwikkeling van hierdie bitterheid het oor die jare heen baie aandag geniet, maar geen definitiewe verklaring is nog daarvoor gevind nie. Die eerste doelwit van hierdie studie was om MSB vanaf drie rooidruifvariëteite, nl. Pinotage, Merlot en Cabernet Sauvignon, te isoleer, te kwantifiseer en te identifiseer. Die MSB-populasies op die druiwe van al drie variëteite het gedurende die 2001- en 2002-parsseisoene tussen 102 en 104 kvu/ml gevarieer. Die Cabernet Sauvignon-druiwe het effens hoër getalle as die Pinotage- en Merlot-druiwe gehad. Die MSB-populasies in die Cabernet Sauvignon-, Pinotage- en Merlot-wyne aan die einde van die alkoholiese fermentasie het tussen 102 en 1055 kvu/ml gevarieer. Gedurende 2002 het die MSB-getalle in die wyne waarin appelmelksuurgisting (AMG) aan die gang was tussen 104 en 108 kvu/ml gevarieer. Die geïsoleerde MSB was onderverdeel in die drie metaboliese groepe, met 59% wat behoort aan die fakultatiewe, heterofermentatiewe groep, 26% aan die obligate, heterofermentatiewe groep en 15% aan die obligate, homofermentatiewe groep. Die isolate is geïdentifiseer as Leuconostoc mesenteroides (4), Oenococcus oeni (28), Lactobacillus brevis (15), Lactobacillus hi/gardii (15, Lactobacillus p/antarum (98), Lactobacillus pentosus (12), Lactobacillus parap/antarum (3), Lactobacillus paracasei (28), Pediococcus acidi/actici (2) en Pediococcus spp. (35) deur middel van spes iespesifieke inleiers. Die mees algemeen geïsoleerde spesies was Lb. p/antarum, gevolg deur Pediococcus spp. Die resultate impliseer dat Pinotage 'n meer uiteenlopende MSB-populasie in vergelyking met Merlot en Cabernet Sauvignon dra. Die tweede doelwit van hierdie studie was om die teenwoordigheid van die gliseroldehidratase-geen in die MSB-isolate deur middel van die GD1- en GD2- inleiers te bepaal. Ses-en-twintig isolate was positief, nl. Lb. p/antarum (15), Lb. pentosus (1), Lb. hi/gard;; (5), Lb. paracasei (2), Lb. brevis (2) en 'n Pediococcus spp. (1). 'n Interessante resultaat was dat 62% van hierdie isolate vanaf Pinotage geïsoleer is. Die isolate was almal in staat om meer as 90% van die gliserol te gebruik en geen noemenswaardige verskille is tussen die isolate waargeneem nie. Die GD-bevattende isolate het verskillende grade van inhibisie teenoor Grampositiewe en Gram-negatiewe bakterieë getoon, en die resultate impliseer dat hierdie inhiberende aktiwiteit dieselfde is as dié van reuterin wat deur Lb. reuteri geproduseer word. Hierdie studie kan dus die basis vorm vir die ontrafeling van die oorsake van bitterheid in rooiwyne. Deur die resultate van hierdie studie met analitiese, sensoriese en molekulêre data te kombineer, kan die wynbedryf voorsien word van waardevolle metodes om die voorkoms van bitterheid mee te bekamp.

Thesis (MSc)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Winemaking is an age-old tradition that dates back to as early as 6000 BC. In our modern era there are several insects and microorganisms that pose a threat to the grapevine, the environment and the final wine product. Farmers and winemakers are becoming aware of the threat and the fight against disease, spoilage and/or pathogenic microorganisms is on the rise. Currently, the natural environment is being altered through rural developments, pollution and disaster, which in turn is responsible for altering the natural micro flora. The result is a harsh battle between man and microorganism. The weapon used often against microorganisms is chemical preservatives, such as sulphur dioxide. These chemical preservatives change the nutritional value, quality and wholesomeness of the wine. Chemical preservatives suppress the quality of the wine with a reduction in wine consumption by the consumers. Until the 18th century, wine was regarded as a safe drink and prescribed by doctors. In the zo" century alcohol consumption became the focus point of some health campaigners. Medical science restored the good name of wine in the 1990s when it came to light that moderate red wine consumption may aid in preventing heart disease and assist in stress management. The only drawback that lowers consumption levels is the use of chemical preservatives. It is of utmost importance to place the focus on health issues and the development of natural preservation methods that are environmentally friendly and contributes to the overall wholesomeness of the wine. Due to these demands, the scientific community placed the focus of research projects on the development and enhancement of biopreservation methods, in order to minimise chemical preservation use. One of the most promising biocontrol agents is bacteriocins. These proteinaceous molecules produced by various lactic acid bacteria exert antimicrobial activity towards closely related organism. Research has shown that bacteriocins may aid in the prevention of wine-spoilage and enhance natural preservation techniques. Most of the research on biopreservation in food and beverages has been performed on the bacteriocins of LAB. No evidence could be found that indicated bacteriocin production by wine isolated LAB in South Africa. This study is therefore, of utmost importance and is considered to be novel pioneering work for the South African wine industry. The main objective of this study was to screen wine isolated LAB for the production of antimicrobial and/or antifungal compounds. This was followed by the isolation and characterisation of the produced bacteriocins. This study forms part of a greater project that focuses on wine preservation, under the auspices of the Institute for Wine Biotechnology.The research results in this study indicated the production of bacteriocins by wine isolated LAB of South African origin. It was found that numerous isolates exerted antimicrobial activity towards other wine associated LAB. The most predominant species that gave the highest activity was Lactobacillus brevis and Lactobacillus paracasei. Experimental results indicated that the bacteriocins produced by these two species were thermo-stable and active over a wide pH range, including the temperatures and pH values that reign in the South African wine environment. The antimicrobial activity was lost after treatment with proteolytic enzymes, such as proteinase K and lysozyme. The size, production and growth kinetic curves of the bacteriocins under investigation showed similar results that are comparable to other findings in the literature. Antifungal activity was detected against Botryfis cinerea that indicated limited inhibitory activity towards spore germination, but had no effect on hyphal growth. This study provides novel information regarding bacteriocin production by LAB isolated from the South African wine industry. The results indicate the suitability of these bacteriocins as possible biopreservatives in the wine environment. The proposed results obtained in this study will aid in the development of bacteriocinproducing, tailored made wine yeast or LAB that may in future, play vital roles in the winemaking process. / AFRIKAANSE OPSOMMING: Wynmaak is 'n eeu oue tradisie wat terugdateer tot so vroeg soos 6000 jaar v.C. In ons moderne eeu is daar verskeie insekte en mikro-organismes wat In bedreiging vir die wingerdstok, asook die omgewing en die finale wynproduk inhou. Boere en wynmakers word al hoe meer bewus van hierdie bedreiging, terwyl die stryd teen siektes, bederf en/of patogene mikro-organismes ook aan die toeneem is. Tans word die natuurlike omgewing deur landelike ontwikkeling, besoedeling en natuurlike rampe verander, wat op sy beurt weer verantwoordelik is vir die verandering van mikroflora. Die gevolg is 'n harde stryd tussen die mens en mikro-organismes. Die wapen wat gereeld ingespan word in die stryd teen mikro-organismes, is chemiese preserveermiddels, soos swaweidioksied. Hierdie chemiese preserveermiddels verander die voedingswaarde, kwaliteit en die voedsaamheid van die wyn. Dit onderdruk ook die gehalte van wyn, wat meebring dat minder wyn deur die verbruiker gedrink word. Tot en met die agtiende eeu is wyn deur dokters as 'n veilige drankie voorgeskryf. In die twintigste eeu het alkoholverbruik die fokuspunt van gesondheidskamvegters geword. In die 1990's het die mediese wetenskap wyn se goeie naam in ere herstel toe dit aan die lig gekom het dat In matige verbruik van rooiwyn moontlik hartsiektes kan voorkom en help om stres te beheer. Die enigste nadelige faktor wat verbruikersvlakke verlaag, is die gebruik van chemiese preserveermiddels. Dit is uiters noodsaaklik om die fokus op gesondheidskwessies te plaas en die ontwikkeling van natuurlike preserveermetodes wat omgewingsvriendelik is en tot die algehele voedsaamheid van wyn bydra. As gevolg van hierdie eise het wetenskaplikes die fokus geplaas op navorsingsprojekte vir die ontwikkeling en verbetering van biopreserveringsmetodes met die doelom die gebruik van chemiese preserveermiddels te verminder. Een van die belowendste biokontrolemiddels is bakteriosiene. Hierdie proteïenagtige molekule word deur verskeie melksuurbakterieë vervaardig en oefen anti-mikrobiese aktiwiteit teenoor nabyverwante organismes uit. Navorsing het getoon dat bakteriosiene moontlik kan help in die voorkoming van wynbederf en natuurlike preserveertegnieke kan verbeter. Die meeste van die navorsing op biopreservering in voedsel en drank is op die bakteriosiene van melksuurbakterieë uitgevoer. Geen bewys kon gevind word in Suid Afrika wat bakteriosienproduksie deur wyn-geïsoleerde melksuurbakterieë aangedui het nie. Hierdie studie is daarom baie belangrik en word as baanbreker werk vir die Suid Afrikaanse wynbedryf beskou. Die hoofdoel van hierdie studie was om wyn-geïsoleerde melksuurbakterieë vir die produksie van anti-mikrobiese en/of anti-fungiese substanse te toets. Dit is gevolg deur die isolasie en karakterisering van die geproduseerde bakteriosiene. Hierdie studie maak deel uit van 'n groter projek wat fokus op wynpreservering en wat onder leiding van die Instituut van Wynbiotegnologie uitgevoer word. Navorsingsresultate van hierdie studie dui op die produksie van bakteriosiene deur wyn-geïsoleerde melksuurbakterieë van Suid Afrikaanse oorsrong. Daar is gevind dat verskeie isolate anti-mikrobiese aktiwiteit teenoor ander wynverwante malksuurbakterieë uitgeoefen het. Die oorheersende spesie wat die hoogste aktiwiteit getoon het, was Lactobacillus brevis en Lactobacillus paracasei. Eksperimentele uitslae dui daarop dat die bakteriosiene wat deur hierdie twee spesies geproduseer word, termostabiel en aktief is oor 'n wye pH reeks, insluitende die temperature en pH-waardes wat in die Suid Afrikaanse wynomgewing voorkom. Die anti-mikrobiese aktiwiteit het verlore gegaan na behandeling met proteolitiese ensieme soos proteïnase K. Die groote, produksie en groeikinetika kurwes van die bakteriosiene wat ondersoek is, toon vergelykbare resultate met ander bevindings in die literatuur. Anti-fungiese aktiwiteit is opgemerk teen Botrytis cinerea, wat beperkte inhiberende aktiwiteit ten opsigte van spoorontkieming aangedui het, maar geen effek op hifegroei gehad nie. Hierdie studie verskaf nuwe inligting aangaande bakteriosienproduksie deur melksuurbakterieë wat van die Suid Afrikaanse wynomgewing geïsoleer is. Die resultate dui op die geskiktheid van hierdie bakteriosiene as moontlike biopreserveermiddels in die wynbedryf. Die voorgestelde resultate deur hierdie studie verkry sal help in die ontwikkeling van bakteriosien produserende, spesifiek vervaardigse wyngis of melksuurbakterieë, wat in die toekoms 'n baie belangrike rol in die wynmaakproses sal speel.

Thesis (MScAgric)--Stellenbosch University, 1978. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Dissertation (PhD Agric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the malolactic enzyme (MLE). The consequence of this conversion is a decrease in total acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of the bacteria the organoleptic profile of the wine is modified. In some wines MLF is considered as spoilage, especially in warm viticultural regions with grapes containing less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine colour, and biogenic amines may be produced. To induce MLF we provided s. cerevisiae with the enzymatic activities required for MLF, which is then conducted by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene (mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and expressed in S. cerevisiae. The activity of this enzyme was compared to two other malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni La11, expressed in the same yeast strain. All three recombinant strains of S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion rate and a significant lower final L-Iactate level were observed with the yeast expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the main organism responsible for MLF, could be safely repressed with a ribosomaly synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB 1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C, and ethanol or S02 does not affect its activity. The peptide was purified to homogeneity and sequence analysis suggests that the peptide is a member of the lantibiotic family of bacteriocins. The molecular mass was estimated by mass spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall biosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range, optimal activity was recorded at pH 5.0. The petide is, however, more stable between pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0. Pediocin PD-1 provides a safer biological alternative than chemical preservatives such as S02. / AFRIKAANSE OPSOMMING: Appelmelksuurgisting (AMG) word deur sekere melksuurbakterieë (MSB) uitgevoer en verwys na die dekarboksilering van L-malaat na L-Iaktaat, 'n reaksie gekataliseer deur die appelmelksuurensiem (AME). AMG verlaag die suurvlakke in wyn, speel 'n rol in mikrobiologiese stabiliteit, en verander die organoleptiese profiel van die wyn. In sommige wyne word AMG beskou as bederf, veral in warm wynbou streke met minder malaat in druiwe. AMG kan ongewenste organoleptiese veranderinge teweeg bring, die wyn se kleur beinvloed, en tot die produksie van biogene amiene lei. Vir die bevordering van AMG het ons S. eerevisiae met die ensiematiese aktiwiteit benodig vir AMG voorsien wat dan veilig deur die gis tydens alkoholiese fermentasie uitgevoer word. 'n AME-koderende geen (mIeD) is uit Pedioeoeeus damnosus NCFB 1832 gekloneer, gekarakteriseer en in S. Cerevisiae uitgedruk. Die aktiwiteit van die ensiem is vervolgens vergelyk met die aktiwitet van twee ander AME gene, mIeS van Laetoeoeeus laetis MG1363 en mleA van Oenoeoeeus oeni Lal1, uitgedruk in dieselfde gisras. AI drie rekombinante gisrasse het L-malaat binne die bestek van drie dae na L-Iaktaat omgeskakel en die finale L-malaat vlakke was minder as 0.3 gIL. Die tempo van omkakeling was egter laer in die gis wat die mIeD geen uitdruk en die finale L-Iaktaat vlakke was veel laer. Om AMG te inhibeer is die groei van O. oeni, die organisme hoofsaaklik verantwoordelik vir AMG, onderdruk deur die byvoeging van 'n ribosomaal gesintetiseerde antimikrobiese peptied, pediocin PD-1, geproduseer deur P. damnosus NCFB 1832. Gisgroei is nie geaffekteer nie. Pediocin PD-1 is stabiel in wyn by temperature wat wissel tussen 4°C en 100°C, en die aktiwiteit van die peptied word nie geaffekteer deur ethanol of S02 nie. Die peptied is gesuiwer volgens In eenvoudige metode wat amoniumsulfaat-presipitasie en katioon uitruilings-ehromatografie insluit. Aminosuur volgorde bepaling van gesuiwerde peptied dui daarop dat pediocin PD-1 tot die lantibiotiese familie van bakteriosiene behoort. Die molekulêre massa van die peptied, soos bepaal deur massa spektroskopie, is 2866.7 ± 0.4 Da. Pediocin PD-1 vorm porieë in selmembrane van sensitiewe selle soos aangedui deur die uitvloei van K+vanuit O. oeni selle. Die peptied kombineer hierdie aksie met die inhibisie van selwand biosintese wat lei tot sel lise. Verlies van sellulêre K+verminder by laer temperature, waarskynlik as gevolg van verandering in die lipied- en protein inhoud van die sitoplasmiese membraan. Alhoewel die peptied aktief is oor 'n breë pH grens, is die antimikrobiese aksie optimaal by pH 5.0. Die peptied is meer stabiel tussen pH 2.0 en 5.0 en toon die beste stabiliteit tussen pH 3.0 en 4.0. Peiocin PD-1 is 'n veilige biologiese alternatief vir chemiese preserveermiddels soos S02.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes the use of a gastro-intestinal model to screen lactic acid bacteria isolated from the gastro-intestinal tract of post-weaned piglets (raised on six different diets) for probiotic properties. Intestinal bacteria were isolated from ,the stomach, duodenum, jejunum, caecum, ileum and colon. The highest cell numbers (6 x 107 cfulg) were isolated from the ileum. No significant differences in viable cell counts were recorded for piglets raised on the six diets. Isolates with the best overall probiotic properties were identified as members of Lactobacillus salivarius and Lactobacillus fermentum. The two strains selected for further studies were Lactobacillus plantarum 423 (originally isolated from sorghum beer) and Lactobacillus salivarius 241 (isolated from pig intestine). Enterococcus faecalis FAIR E 92 was originally isolated from pig intestine and was included in this study as a non-pathogenic challenge strain. L. plantarum 423 produces a bacteriocin plantaricin 423, active against E. faecalis FAIRE 92. L. plan/arum 423 and L. salivarius 241 were included in the gastro-intestinal model and their adhesion to the mucus of porcine ileum studied with fluorescent-in-si/u-hybridization (FISH). A decrease in viable cell numbers of L. plan/arum 423 was recorded in the duodenum, jejunum and ileum in the presence of bile and pancreatic juice. However, higher cell numbers were recorded in the caecum and anterior colon, which suggested that strain 423 recovered from these stress factors. Plantaricin 423 was detected for up to 28 hours in the duodenum, jejunum, ileum and middle colon. Lower cell numbers (one log unit) of L. salivarius 241 were recorded in the gastro-intestinal model over seven days, compared to strain 423. Piglets of one, 14 and 28-days-old were dosed with L. plan/arum 423 and L. salivarius 241, separately and in combination (1: 1). In a separate experiment, 14-day-old piglets were challenged twice with E. faecalis FAIRE 92, followed by dosage with strains 423 and 241. New-borne piglets dosed with L. plantarum 423 gained more weight (4 kg over 19 days) compared to piglets dosed with L. salivarius 241 (2.2 kg over 19 days), or a combination of the two strains (2 kg over 19 days). Piglets of 14 and 28-days-old, on the other hand, gained more weight when dosed with a combination of strains 423 and 241. The cell numbers of E. faecalis FAIR E 92 and other enterococci decreased drastically (two log units) when the piglets were dosed with the latter two strains. Overall, piglets of various ages reacted differently when administered L. plantarum 423 and L. salivarius 241, separately or in combination. Fluorescent-in-situ-hybridization (FISH) was used to study the in vivo adhesion of L. plantarum and L. salivarius to mucus in the stomach, duodenum, jejunum, ileum, caecum and colon. The highest number of L. plantarum cells was recorded in the ileum, whereas L. salivarius favoured adhesion to the duodenum. A decrease in cell numbers of E. faecalis in the ileum mucus was recorded when a combination of the probiotic strains 423 and 241 was administered. This study provided a reliable estimation of the presence and/or adhesion of L. plantarum and L. salivarius to various parts of the porcine gastro-intestinal tract, without the use of expensive cultivation techniques. Insight was gained into the co-evolution existing between probiotic bacteria and the porcine gastro-intestinal tract, emphasizing the use of gastro-intestinal models to study the dynamics of the gastro-intestinal tract. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf die gebruik van 'n gastro-intestinale model, om melksuurbakterieë wat geïsoleer is uit die spysverteringskanaal (SVK) van reeds gespeende varkies (gevoed op ses verskillende diëte) vir probiotiese eienskappe te toets. Ingewandsbakterieë is uit die maag, duodenum, jejunum, caecum, ileum en kolon geïsoleer. Die hoogste aantal selle (6 x 107 kve/g) is geïsoleer uit die ileum. Geen betekenisvolle verskille in lewensvatbare seltellings, vir varkies gevoed op ses verskillende voere is aangeteken nie. Isolate met die beste algehele probiotiese eienskappe is as Lactobacillus salivarius en Lactobacillus fermentum geïdentifiseer. Vir verdere studie is twee isolate Lactobacillus plantarum (oorspronklik uit sorghum-bier geïsoleer) en Lactobacillus salivarius (uit die varkdermkanaal geïsoleer) geselekteer. Enterococcus faecalis FAIRE 92, oorspronklik uit die varkdermkanaal geïsoleer, is in hierdie studie as 'n nie-patogeniese indikator gebruik. L. plantarum 423 produseer 'n bakteriosien plantarisien 423 wat aktief is teen E. faecalis FAIR E92. L. plantarum 423 en L. sa/ivarius 241 is ingesluit in die gastro-intestinale model, en vashegting van die bakterieë aan die mukus van vark-ileum is met fluoresensie-in-si/uhibridisasie (FISH) bestudeer. 'n Afname in lewende selgetalle van L. plan/arum 423 in die duodenum, jejunum en ileum is aangetoon in reaksie tot die byvoeging van gal en pankreatiese sappe. Hoër selgetalle is nietemin aangeteken in die caecum en voorste gedeelte van die kolon, wat 'n aanduiding gee dat isolaat 423, ten spyte van hierdie stres-faktore, oorleef. Plantaricin 423 is vir 'n tydperk (28 uur) in die duodenum, jejunum, ileum en sentrale kolon gevind. Laer selgetalle (een logaritmiese eenheid) van L. salivarius 241 is in die gastro-intestinale modeloor 'n tydperk van sewe dae aangetoon, in vergelyking met isolaat 423. Een, 14 en 28 dag oud varkies is met L. plantarum 423 en L. salivarius 241 (afsonderlik en in kombinasie 1:1) twee keer gedaag met E. faecalis FAIR E 92, opgevolg met dosering van 423 en 241. Pasgebore varkies het die hoogste gewigstoename getoon (4 kg oor 19 dae) na dosering met L. plantarum 423 in vergelyking met varkies gedoseer met L. salivarius 241 (2.2 kg oor 19 dae) of 'n kombinasie van die twee isolate (2 kg oor 19 dae). Daarenteen het veertien- en 28 dag oud varkies beter gewigstoename getoon na dosering met 'n kombinasie van isolate 423 en 241. Die selgetalle van E. faecalis FAIRE 92 en ander enterococci het drasties afgeneem (twee logaritmiese eenhede) nadat die varkies met laasgenoemde twee isolate gedoseer is. Varkies van onderskeie ouderdom het verskillend gereageer na dosering met L. plantarum 423 en L. salivarius 241 afsonderlik of in kombinasie. Fluoresensie-in-situ-hibridisasie (FISH) is gebruik om die in vivo vashegting van L plantarum en L. salivarius tot die vark mukus in die maag, duodenum, jejunum, ileum, caecum en kolon te bestudeer. Die hoogste telling van L. plantarum selle is aangeteken in die ileum, terwyl L. salivarius aanhegting tot die duodenum verkies het. 'n Afname in seltellings van E. faecalis in die ileum mukus was aangeteken na toediening met 'n kombinasie van probiotiese isolate 423 en 241. Hierdie studie het 'n betroubare bepaling van die voorkoms en/ofvashegting van L. plantarum en L. sa/ivarius isolate in verskeie gedeeltes van die varkspysverteringskanaal voorsien, sonder die hulp van duur kwekings tegnieke. Probiotiese bakterieë is in 'n gastro-intestinale model, wat die natuurlike omgewing verteenwoordig, bestudeer. Insig oor die ko-evolusie tussen probiotiese bakterieë en die SVK van die vark is verkry. Die gebruik van 'n gastro-intestinale model om die dinamika van die SVK te bestudeer is met hierdie studie beklemtoon.