Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The winemaking process involves a number of microorganisms, each with its own role. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are Gram-positive bacteria associated with must and wine and perform the malolactic fermentation (MLF), while the acetic acid bacteria (AAB) are Gram-negative bacteria converting ethanol to acetic acid. These microorganisms are present in the cellar and fermentation tanks and can be seen either as beneficial or as wine spoilage microorganisms because, under certain circumstances, they affect the wine quality if they should grow in the wine or must. Strict measures need to be implemented in the cellar during the winemaking process to ensure microbiological stability. This can be achieved through good microbiological practices and, additionally, chemical preservatives. Sulphur dioxide (S02) is widely used as the primary preservative in winemaking. However, consumer resistance has been building up against the use of chemical preservatives, due to the possible health risks and a decrease in nutritional value and sensorial quality of certain foods and beverages. Biopreservation as an alternative to the traditionally-used chemical preservation is a new approach and has been attracting much attention. This implies the use of the natural microflora and/or their antibacterial products, such as bacteriocins and bacteriolytic enzymes (e.g. lysozyme). Bacteriocins from LAB are proteins or protein complexes, produced by Gram-positive bacteria, with antibacterial activity against closely-related Gram-positive species. Lysozyme occurs in substances such as hen egg white and has lytic activity against Gram-positive bacteria. ' The bacteriocins nisin, of the class I lantibiotics, and pediocin PA-1 and leucocin BTA 11a, of the class lIa Listeria-active bacteriocins, have been investigated for the biopreservation of wine. Nisin, however, is the only bacteriocin that has been approved for use as a preservative, while pediocin is likely to follow in approval. Lysozyme has been approved for use in winemaking by the Office International de la Vigne et du Vin (OIV). The main objectives of this study were to determine whether these substances showed any antimicrobial action against wine-associated microorganisms, namely LAB, AAB and yeasts. The stability and suitability of the bacteriocins and lysozyme as antimicrobials in wine was researched, especially when used in combination. Possible synergistic or antagonistic interactions between the bacteriocins were also investigated by means of the microtitre broth dilution method and scanning electron microscopy, as well as at what concentration and combinations the bacteriocins were most effective against increasing LAB concentrations. It was found that nisin, pediocin and leucocin were effective to varying degrees against a test panel of LAB type and reference strains, as well as wine isolates. Nisin repeatedly had the highest level of inhibition against all the LAB tested, followed by pediocin and leucocin. There was no inhibition of the wine-associated AAB and yeasts tested. Pediocin stability was evaluated in simulated wine must and proved to be stable for at least 20 days, without being affected by the sulphur or alcohol content. A low pH, however, led to a more rapid decrease in activity. The same was found for nisin and leucocin in other studies. Combinations of bacteriocins at increasing concentrations were evaluated against increasing concentrations of a LAB wine isolate. When used in pairs (namely, nisinleucocin, nisin-pediocin and pediocin-Ieucocin), the combinations were most effective against lower concentrations of bacteria, namely 102 and 104 cfu/ml. At lower concentrations, the pairs of bacteriocins were much less effective against the higher bacterial concentrations of 106 and 108 cfu/ml. Leucocin-pediocin combinations were the least effective, while nisin-Ieucocin combinations were marginally more effective than the nisin-pediocin combinations. The most pronounced effect was observed when all three the bacteriocins were used together. Combinations of bacteriocins had no inhibitory effect against AAB. Pediocin and lysozyme was used in combination against the same wine isolate, but no conclusive conclusions could be drawn in this experiment. __ Scanning electron microscopy was used to investigate any disturbances in cell morphology when bacteriocins were added to LAB. The above-mentioned LAB was subjected to bacteriocins used singularly and also in combinations of equal amounts of bacteriocins. The action of the bacteriocins led to major disturbances in cell morphology. Once again, the combination of leucocin-pediocin was the least effective, even less so than when the single bacteriocins were used. The nisin-pediocin and nisin-Ieucocin combinations seemed to be more effective in causing cell disturbances and perturbations. The microtitre broth dilution methodwas used to further characterise the nature of the interaction of the pairs of bacteriocins. This test showed clearly that the bacteriocins had definite interactions. By adding one bacteriocin to varying concentrations of another bacteriocin, the inhibitory action of the second bacteriocin was affected, either increasing or decreasing its effectiveness. The most important factor to consider seems to be the ratio at which the bacteriocins should be used together, leading either to synergism or antagonism, and this also implies a very complex interaction. This project indicated that it is indeed possible to use both bacteriocins and lysozyme in "Vine preservation, both being stable in wine environments and effective against LAB without affecting the yeast fermentation. Bacteriocins could also be used in combination, to broaden the inhibition spectrum, as well as possibly increasing the inhibitory potential of the individual antimicrobials. The underlying interactions in such combinations should be carefully researched, however, when considering using combinations of antimicrobials in food and beverage products. Further attention can also be given to finding biopreservatives against the Gram-negative AAB, as well as to research the interaction of the pairs of bacteriocins over time. Another point to consider would be the engineering of yeasts or bacteria to produce these antibacterial substances in situ as part of their metabolism. / AFRIKAANSE OPSOMMING: Daar is 'n verskeidenheid mikroorganismes in die wynrnaakproses betrokke, elkeen met sy eie rol. Giste is vir die alkoholiese fermentasie verantwoordelik, die Gram-positiewe melksuurbakterieë (MSB) wat in mos en wyn voorkom, is vir die appelmelksuurgisting (AMG) verantwoordelik, terwyl die Gram-negatiewe asynsuurbakterieë (ASB) etanol in asynsuur omskakel. Hierdie mikroorganismes is in die wynkelder en fermentasietenke teenwoordig en kan as óf gunstig óf ongunstig beskou word, afhangende van die toestande waaronder hulle groei en hoe die wyn daardeur beïnvloed word. Om mikrobiologiese stabiliteit in wyn te verseker, moet daar streng higiëniese maatreëls in die kelder toegepas word en word daar ook van addisionele chemiese preserveermiddels gebruik gemaak. Swaweidioksied (S02) word tans algemeen as pnmere preserveermiddel in die wynbedryf gebruik. Weens die moontlike gesondheidsrisiko's wat S02 mag inhou en die moontlike verlaging van die voedingswaarde en sensoriese gehalte waarmee dit in sommige voedsel- en drankprodukte geassosieer word, bou daar tans verbruikersweerstand teen die gebruik daarvan as chemiese preserveermiddelop. Biopreservering is 'n alternatief tot hedendaagse chemiese preservering en het reeds baie belangstelling ontlok. Hierdie metode impliseer die gebruik van die natuurlike mikroflora en/of die antimikrobiese produkte van hierdie rnikroërqanisrnes, soos bakteriosiene en bakteriolitiese ensieme (bv. lisosiem). Bakteriosiene van MSB is proteïene of proteïenkomplekse met antimikrobiese aktiwiteit teen naby-verwante Grampositiewe spesies. Lisosiem kom in produkte soos hoendereierwit voor en het litiese aktiwiteit teen Gram-positiewe bakterieë. Die bakteriosiene nisien, wat tot die klas I lantibiotiese bakteriosiene behoort, en pediosien PA-1 en leukosien B-TA11a, wat tot die klas lIa Listeria-aktiewe bakteriosiene behoort, is as moontlike biopreserveringsagense in wyn ondersoek. Nisien is egter tot op hede die enigste bakteriosien wat amptelik vir gebruik as 'n preserveermiddel in voedsel goedgekeur is, terwyl pediosien moontlik sal volg. Lisosiem is vir gebruik in wynmaak deur die Office International de la Vigne et du Vin (OIV) goedgekeur. Die hoofdoelwitte van hierdie studie was om te bepaal of die bogenoemde stowwe antimikrobiese werking teen wyngeassosieerde mikroorganismes het, soos die ongewenste MSB, ASB en giste. Die stabiliteit en geskiktheid van dié bakteriosiene en lisosiem as antimikrobiese middels in wyn is ook ondersoek, veral wanneer hulle in kombinasie vir preservering gebruik is. 'n Mikrotiterverdunningsboeljon-metode en skanderingselektronmikroskopie is gebruik om moontlike sinergistiese en antagonistiese interaksies tussen bogenoemde bakteriosienpare te ondersoek. Terselfdertyd is die effektiefste konsentrasies en kombinasies van bakteriosiene teen stygende MSB-getalle bepaal. Daar is bevind dat nisien, pediosien en leukosien in verskillende mates teen 'n toetspaneel van MSB tipe- en verwysingsrasse, asook MSB-wynisolate, effektief is. Nisien was herhaaldelik die effektiefste teen dié MSB, gevolg deur pediosien en dan leukosien. Die bakteriosiene was nie teen die wyngeassosieerde ASB of giste wat getoets is, effektief nie. Daar is ook bewys dat pediosien vir tot 20 dae stabiel in 'n gesimuleerde wynomgewing was, sonder dat die alkohol- of die swaweldioksiedkonsentrasie 'n invloed op die aktiwiteit gehad het nie. 'n Lae pH het geblyk om die grootste invloed op die afname in aktiwiteit te hê. Hierdie bevindinge ten opsigte van pediosien het die resultate van nisien en leukosien in ander, soortgelyke ondersoeke, bevestig. Die werking van toenemende konsentrasies van bakteriosienkombinasies (as pare van nisien-Ieukosien, nisien-pediosien, leukosien-pediosien, en al drie saam as nisienpediosien- Ieukosien) teen toenemende getalle van In wyngeïsoleerde MSB is geëvalueer. Wanneer die bakteriosiene in pare gebruik is, was die kombinasies die effektiefste teen laer MSB selgetalle (102 en 104 kfe/ml), terwyl dit baie minder effektief teen hoër selgetalle (106 en 108 kfe/ml) was, veral wanneer lae bakteriosienkonsentrasies gebruik is. Die nisien-Ieukosien kombinasiewas tot 'n geringe mate meer effektief as die nisien-pediosien kombinasie. Die leukosien-pediosien kombinasie het die laagste effektiwiteit van.al die pare bakteriosiene wat gebruik is, getoon. Die sterkste werking is waargeneem toe al drie die bakteriosiene saam teen bogenoemde MSB gebruik is. Die bakteriosien kombinasies het geen effek teen ASB gehad nie. Pediosien en lisosiem is ook in kombinasie teen dieselfde wynisolaat gebruik, maar geen oortuigende afleidings kon van hierdie eksperiment gemaak word nie. Skanderingselektronmikroskopie is gebruik om enige morfologiese verandering in die MSB-wynisolaat waar te neem wanneer bakteriosiene daarby gevoeg is. Dieselfde wynisolaat is weer gebruik en bakteriosiene is by die bakterieë gevoeg, enkelvoudig asook in kombinasies (soos voorheen gebruik) teen gelyke hoeveelhede. Die werking van die bakteriosien het gelei na merkbare veranderinge in selmorfologie, en die kombinasie van pediosien-Ieukosien was weereens die minste effektief. Die mikrotiterverdunningsboeljon-metode is gebruik om die aard van die bakteriosieninteraksies verder te karakteriseer. Die toetse het duidelik aangedui dat die bakteriosiene op mekaar reageer. Deur een bakteriosien tot variërende konsentrasies van 'n ander bakteriosien te voeg, is die inhibitoriese werking van die tweede bakteriosien geaffekteer deurdat die effektiwiteit daarvan toegeneem of afgeneem het. Dit het ook geblyk dat die belangrikste faktor wat hier in ag geneem moet word die verhouding is waarteen die bakteriosiene met mekaar gebruik word, aangesien dit tot óf sinergisme óf antagonisme kan lei. Dft dui op 'n baie komplekse interaksie. Die resultate van hierdie projek het dus daarop gedui dat dit inderdaad moontlik is om beide bakteriosien en lisosiem in wynpreservering te gebruik, aangesien beide nie net stabiel in 'n wynomgewing is nie, maar ook effektief is teen MSB sonder dat die gisfermentasies geaffekteer word. Bakteriosiene kan ook in kombinasie gebruik word om die inhibisie spektrum te verbreed, en om ook moontlik die inhibisiepotensiaal van die individuele peptiede te verhoog. Onderliggende interaksies by sulke kombinasies moet egter sorgvuldig ondersoek word wanneer daar oorweeg word om kombinasies van hierdie antimikrobiese middels in voedsel- en drankprodukte te gebruik. Verder moet daar ook aandag geskenk word om biopreserveermiddels te vind wat ook teen die Gram-negatiewe ASB effektief is, asook aan die aard van die verloop van interaksies van pare van bakteriosiene oor tyd. Nog 'n punt om te oorweeg is die manipulasie van giste of bakterieë omdie antimikrobiese peptiede in situ, as deel van hulle metabolisme, te produseer.

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 (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its aroma and flavour. However, they can also be responsible for many wine spoilage problems that compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl carbamate. Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to reduce SO2 levels due to the increasing health related risks and other factors. All these considerations have increased the interest in research to look for new preservation strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the last decade. Various types of bacteriocins have been identified and characterized. However, there are few reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished wine. The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin production. The isolates originated from South African red wines undergoing spontenous malolactic fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR fingerprinting gave the same profile for the seven strains confirming that they were all identical on genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below 4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results confirmed that all eight strains contained the enterocin P gene from Ent. faecium. The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at 121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci producing bacteriocins have potential applications in various foods and fermented products. The pH tests showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where products are acidified or pH decreases due to natural LAB present. They also have potential applications in oenological process where pH levels are as low as 3 and 4. Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated that the compound was proteinaceous in nature. Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314 was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the control increased exponentially throughout the experiment. In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore have a potential role in wine spoilage control. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings, soos biogeniese amienes en etielkarbamaat, te produseer. Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn. Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in gesondheidsverwante risiko’s en ander faktore. Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor bakteriosiene wat in afgeronde wyn teenwoordig is. Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind. Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was. RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE, het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al agt rasse die enterosien-Pgeen van Ent. faecium bevat. Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het 50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is. Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief gemaak, wat getoon het dat die samestelling proteïenagtig van nature is. Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het. Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera. / TIA, NRF and THRIP