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.

Thesis (MSc (Institute for Wine Biotechnology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The process of winemaking involves a number of microorganisms, contributing both positively and negatively to the final product. Lactic acid bacteria (LAB) are present at all stages of vinification and therefore play a major role in the production of wine, especially red wine. LAB are responsible for malolactic fermentation (MLF), which can be desirable or unwanted depending on the style of wine. LAB can also be responsible for spoilage, and production of off flavours resulting in a decrease in the quality of the finished wine. Spoilage occurs if the wrong species are present at the wrong time and can also occur as a result of spontaneous MLF. It is therefore necessary to control the population of indigenous LAB present in the wine. Plantaricins are bacteriocins produced by Lactobacillus plantarum strains and have the potential to inhibit closely related strains that occupy the same ecological niche. This makes them promising for the control of LAB during the winemaking process. Inhibition of the indigenous LAB microflora could help to prevent the formation of undesirable off-flavours, as well as allowing for control over MLF. The use of plantaricin-producing starter cultures could also lead to a reduction in the amount of sulphur dioxide used in wine. The purpose of this study was to investigate the potential of L. plantarum strains isolated from wine to produce plantaricins under winemaking conditions. This potential was evaluated by investigating the expression of plantaricin genes under winemaking conditions. The first objective was to screen nineteen strains of L. plantarum isolated from South African red wines, as well as a commercial strain; for various genes responsible for the production of plantaricins, including structural, transport and regulatory genes. Results showed that the twenty strains contained at least 16 of the 24 genes (previously reported to be associated with the plantaricin locus for various L. plantarum strains) screened for. Only orfZ123 and orf345 genes yielded no positive results in any of the strains. The second objective was to sequence selected plantaricin genes (plnE, plnF, plnN, plnG and plnB) to determine the variation in nucleotide and amino acid sequences of these genes among the different wine L. plantarum isolates. High homology was found between the nucleotide sequences of the strains and none of the amino acid substitutions in the protein sequences occurred in conserved regions. The nucleotide sequence of plnN was identical in all but one of the strains and similarity of the plnB sequence ranged from 96% to 100%. Similarity of the plnG nucleotide sequence ranged from 99% to 100%. The plnE nucleotide sequence was identical in all but two strains and there were only two groups in terms of nucleotide sequence for plnF, with only two changes between the groups. The third objective was the evaluation of plantaricin production using plate assays mimicking certain wine parameters (pH and ethanol concentration). All twenty strains showed inhibitory activity to varying degrees against a panel of nine indicator microorganisms, including Enterococcus faecalis, Listeria monocytogenes and potential wine spoilage organisms, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides. Addition of 10% ethanol and a low pH of 3.5 decreased both the bacteriocin production as well as the spectrum of activity. Seven of the twenty strains, however, showed good bacteriocin activity under all conditions. The fourth objective was to investigate the expression of two plantaricin structural genes (plnEF and plnJK) and the transporter gene (plnG) under winemaking conditions. Two strains (R1122 and 113.1) were chosen, based on the results from the previous objectives, as starter cultures for MLF in synthetic wine media and Riesling wine. Low wine pH (3.2) and high wine pH (3.8) levels were investigated in conjunction with ethanol concentrations of 0%, 12% and 15%. All three of the genes were expressed to varying degrees depending on the fermentation condition. High ethanol and low pH generally decreased expression of the structural plantaricin genes. The influence on expression of the transporter gene was different, with low pH and presence of ethanol resulting in an increase in gene expression. The genes were also expressed in wine, although at a lower level relative to expression in the synthetic wine media. The presence of sensitive bacteria in the wine seemed to increase expression of the structural genes. Furthermore, expression of the mle gene responsible for MLF was investigated under the same winemaking conditions. Expression was shown to be inducible by malic acid, and negatively affected by the presence of ethanol but positively influenced by a lowering in pH from 3.8 to 3.2. This study confirms that plantaricin genes are expressed under winemaking conditions, which in turn indicates that the plantaricins could be produced under winemaking conditions. This confirms the potential use of these plantaricin-producing strains as starter cultures for MLF with the ability to inhibit indigenous LAB, however, presence of the plantaricin protein in wine still needs to be confirmed. It will also need to be established whether the protein is biologically active and not inhibited by wine-related factors. / AFRIKAANSE OPSOMMING: Die proses van wynmaak bevat 'n verskeidenheid mikroorganismes, wat postiewe en negatiewe bydrae kan lewer tot die finale produk. Melksuurbakterieë is teenwoordig by alle stadiums van wynmaak en speel 'n belangrike rol in die produksie van wyn. Melksuurbakterieë is verantwoordelik vir appelmelksuur gisting (AMG), wat gewens of ongewens kan wees, afhangende van die styl van die wyn. Melksuurbakterieë kan ook verantwoordelik wees vir bederf van wyn, asook die produksie van ongewenste geure wat bydrae tot ʼn toename in die kwaliteit van die wyn. Bederf van wyn kan gebeur as die verkeerde spesies voorkom op die verkeerde tyd en kan ook gebeur as ʼn gevolg van spontane AMG. Dit is dus nodig om die populasie van natuurlike melksuurbakterieë in wyn te beheer. Plantarisiene, geproduseer deur Lactobacillus plantarum wyn-isolate, het die potensiaal om naby verwante stamme se groei te inhibeer wat in dieselfde nis voorkom. Hierdie eienskap maak hul belowend vir die beheer van melksuurbakterieë se groei gedurende die wynmaakproses. Inhibering van die natuurlike mikroflora kan help om die vorming van ongewenste geure te verhoed, sowel as om AMG te beheer. Die gebruik van aanvangskulture, wat plantarisiene kan produseer, kan lei tot ’n vermindering in die gebruik van swaweldioksied in die wynindustrie. Die doel van hierdie studie was om die potensiaal van L. plantarum stamme, geïsoleer vanuit wyn, te ondersoek vir hul vermoë om plantaricins te produseer in toestande wat die wynmaakproses naboots. Die potensiaal was ondersoek deur te kyk na die uitdrukking van plantarisien-produserende gene onder wynmaak toestande. Die eerste objektief was om die 19 L. plantarum stamme, geïsoleer vanuit Suid-Afrikaanse rooi wyne, asook n kommersiele stam, te ondersoek vir die teenwoordigheid van verskeie gene wat verantwoordelik is vir die produksie van plantarisiene, sowel as strukturele, transporter en regulerende gene. Al twintig van hierdie stamme het ten minste 16 uit die 24 gene bevat waarvoor ondersoek was. OrfZ123 en orf345 het egter geen positiewe resultate opgelewer in enige van die stamme nie. Die tweede objektief was om die DNA-volgorde te bepaal van spesifieke gene (plnE, plnF, plnN, plnG, sowel as plnB) en sodoende die variasie in nukleotied en aminosuur volgorde van hierdie gene in die verskillende L. plantarum wyn-isolate te bepaal. Hoë vlakke van homologie was gevind en geen van die aminosuur veranderings het in behoue gebiede plaasgevind nie. Die nukleotied volgorde van plnN was identies in al die stamme, behalwe vir een, en die ooreenkomste tussen die plnB volgorde het varieër van 96% tot 100%. Die ooreenkomste tussen die plnG nukleotied volgorde het varieër van 99% to 100%. Die plnE nukleotied volgorde was identies in al die stamme, behalwe vir twee, en daar was net twee groepe in terme van nukleotied volgorde vir plnF, met net twee veranderinge tussen die groepe. Die derde objektief was om die vermoë van die stamme om plantaricins the produseer, deur gebruik te maak van plaat assays, onder verskillende wyntoestande te ondersoek. Die twinting stamme het verskillende vlakke van inhibering teenoor die nege toets-organismes getoon, wat Enterococcus faecalis, Listeria monocytogenes sowel as potensiele wyn bederf organismes, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides insluit. Die byvoeging van 10% etanol en ’n lae pH van 3.5, het beide bakteriosien produksie inhibeer, sowel as die spektrum van aktiwiteit verminder. Sewe van die stamme het egter steeds goeie aktiwiteit getoon onder al die kondisies wat getoets was. Die vierde objektief was om die uitdrukking van twee plantaricin strukturele gene (plnEF en plnJK), sowel as die transporter geen (plnG) onder wynmaak omstandighede te ondersoek. Twee stamme (R1122 en 113.1) was gekies as aanvangskulture vir AMG in sintesiese wyn media, sowel as Riesling wyn. Hierdie twee stamme was gekies op grond van die resultate wat van die vorige objektiewe verkry was. Lae wyn pH (3.2) en hoë wyn pH (3.8) was ondersoek in samewerking met verskillende etanol konsentrasies wat 0%, 12% en 15% etanol insluit. Al drie hierdie gene was uitgedruk teen verskillende vlakke, afhangende van die verskeie fermentasie kondisies. Hoë etanol en lae pH lei oor die algemeen tot ʼn toename in uitdrukking van die strukturele plantarisien gene. Die invloed op uitdrukking van die transporter geen was verskillend, want lae pH en die teenwoordigheid van etanol het gelei tot ʼn verhoging in geen uitdrukking. Die gene was uitegdruk in wyn, maar was teen laer vlakke relatief tot uitdrukking in die sintetiese wyn media. Dit blyk dat die teenwoordigheid van sensitiewe bakterieë in die wyn tot ‘n hoër uitdrukking van die strukturele gene lei. Die uitdrukking van die mle geen, verantwoordelik vir AMG, was ook onder dieselfde wynmaak kondisies ondersoek. Die uitdrukking was geïnduseer deur appelsuur, negatief beïnvloed deur die teenwoordigheid van etanol, maar positief beïnvloed deur ’n verlaging in pH van 3.8 tot 3.2. Hierdie studie toon dat plantaricin gene uitegedruk word onder wynmaak toestande en dat plantaricins moontlik onder hierdie toestande geproduseer kan word. Die potensiaal van hierdie stamme word getoon om as aanvangskulture gebruik te word vir AMG, om sodoende die groei van natuurlike melksuur bakterieë te inhibeer. Die teenwoordigheid van die plantarisien peptied in die wyn moet egter nog bewys word. Daar sal ook vasgestel moet word of die peptied biologies aktief is en nie deur wynverwante faktore geïnhibeer word nie.

Les Escherichia coli producteurs de Shiga-Toxines (STEC) sont responsables de maladies humaines sévères. Les ruminants sont considérés comme étant leur principal réservoir. La dissémination des STEC au sein des élevages est liée en partie à l’alimentation des animaux et donc potentiellement à l’ingestion d’ensilages contaminés. Les bactéries lactiques peuvent être employées comme agents technologiques ou dans des stratégies de bio-protection. Sur le plan de l’ensilage, elles jouent un rôle de préservation mais peuvent également représenter une barrière à la survie de pathogènes comme les STEC. Ce travail a permis de sélectionner des bactéries lactiques inhibitrices de la croissance de divers sérogroupes de STEC. Les études de compétitions ont mis en évidence un phénomène bactéricide sur certaines souches, dont le mécanisme reste encore non élucidé. Le potentiel inhibiteur des bactéries lactiques sélectionnées a été testé indépendamment dans des ensilages de maïs contaminés à différentes étapes de leur réalisation : à la mise en silos, à l’ouverture ou après une période d’exposition aérobie. En cas de contamination à la mise en silos, les souches de STEC testées n’ont pas survécu dans des ensilages correctement menés. Une souche de Ln. mesenteroides a permis de limiter la survie des souches de STEC dans les ensilages contaminés à l’ouverture. Cependant, après 144h d’aération, aucun additif n’a montré d’effet protecteur avéré. Le contrôle de l’alimentation animale afin de limiter l’entrée des STEC dans le cycle épidémiologique pourrait donc passer par l’emploi de bactéries lactiques ; sans négliger cependant les Bonnes Pratiques nécessaires à la réalisation de l’ensilage. / Shiga-toxin producing Escherichia coli (STEC) are responsible for severe human diseases. Cattle are considered as the main reservoir of this pathogen. STEC dissemination in farm environment is linked to cattle feed and potentially to ingestion of contaminated silage. Lactic Acid Bacteria (LAB) could be employed as starters in fermentations or in strategies of bioprotection. In silage, LAB play a preservative role and could also represent a barrier for the survival of pathogenic bacteria such as STEC. The selection of LAB strains, able to inhibit the growth of several serogroups of STEC strains, was performed in this study. Competitions assays have shown a bactericidal effect on some STEC strains, but reasons of this phenomenon remain unclear. Inhibiting potential of the selected LAB strains was tested independently in corn silages contaminated at different steps of their realizations : at ensiling, at opening or after aerobic exposure. In case of contamination at ensiling, STEC strains tested did not survive in well-made silages. A Ln. mesenteroides strain allowed the limitation of the STEC strains survival in silage contaminated at opening. However, after 144 h of aerobic exposure, no inoculant showed any protective effect. Control of cattle feed, in order to limit STEC entry in their epidemiological cycle, could be reached through LAB utilization ; however, Good Manufacturing Practices involved in silage making should not be omitted.

Depuis quelques années, les probiotiques sont devenus un front de science très actif. La majorité des bactéries probiotiques actuellement sur le marché sont des bactéries lactiques. L’identification de nouvelles souches bactériennes aux propriétés probiotiques est l’objectif principal de ces travaux de thèse, et s’inscrivent dans le cadre du projet Probiotique en partenariat avec deux groupes industriels et le pôle de compétitivité VITAGORA. / In recent years, probiotics market is boosting. Most probiotic bacteria on the market today are lactic acid bacteria. The identification of novel bacterial strains with probiotic properties is the main objective of this thesis, which is part of the Probiotic project in partnership with two industry groups and the network innovation VITAGORA.

Call number: LD2668 .T4 1986 T39 / Master of Science / Animal Sciences and Industry