Successfully inducing malolactic fermentation in the production of grape wines can be challenging, especially in wines after finishing alcoholic fermentation with limited energy sources, low pH values and high ethanol concentrations. In this thesis, the kinetics of several chemicals of enological relevance were studied in a white wine (Chardonnay) and a red wine (Cab Franc) vinified by traditional, consecutive alcoholic (AF) and malolactic fermentations (MLF), and simultaneous AF/MLF, where bacteria were co-inoculated with yeast. The Chardonnay must was adjusted to four pH values (3.20, 3.35, 3.50 or 3.65), the cab Franc was kept as original pH value (3.56) and the concentrations of sugars, organic acids as well as acetaldehyde were followed throughout the fermentations. For Chardonnay the degradation of glucose and fructose was slower at the lowest must pH value (3.20) and independent from the time of bacterial inoculation. In all cases, malolactic conversion was faster after yeast-bacterial co-inoculation and was completed in simultaneous treatments at pH values of 3.35-3.65, and consecutive treatments at pH 3.50 and 3.65. No statistically significant difference was observed among the final acetic acid concentration, in all inoculation and pH treatments. For Cab Franc, it confirmed that co-inoculation shortened the fermentation periods while having minor effects on other parameters. Overall, simultaneous AF/MLF allowed for greatly reduced fermentation times, while the must pH remained a strong factor for fermentation success and determined the final concentration of various wine components. The time of inoculation influenced formation and degradation kinetics of organic acids and acetaldehyde significantly.

Phenolics are critical to the sensory attributes and health benefits of hard cider due to their contribution to the flavor, mouthfeel, and antioxidant activity. With the increase in demand for cider, the use of dessert apple varieties has become more common leaving ciders lacking in phenolics. However, a promising method to increase their phenolic content is through maceration with apple pomace. This study evaluated the effect maceration with apple pomace during cider fermentation on the extraction of phenolic compounds, as well as its effect on the sensory properties of the final product. For this study, ciders were fermented with 0% (control), 20%, 35%, and 50% of the average pomace created during production of the apple must. After fermentation, the ciders were analyzed for acid content, total phenolic content (TPC), alcohol by volume (ABV), color, volatile profile, and sensory properties. The ciders fermented with apple pomace went through malolactic fermentation. During the maceration phenolics were extracted which resulted in an increase in phenolic content in cider. Compared to the control, the treatment ciders were also seen to have higher ABV and TPC, lower acidity, and a different aromatic profile: decreased acetaldehyde and increased ethyl acetate, isoamyl alcohol, and phenylethyl alcohol. The color measurements suggests that treatment ciders were lighter, with a higher red and yellow color compared to the control cider. The sensory analysis revealed the treatment ciders were perceived as less acidic and astringent, but more bitter than the controls. This study shows the addition of apple pomace in its native state increases total phenolic content and could be favorable to produce well rounded ciders. This study shows maceration is a promising technique for increasing phenolic content in ciders.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Wine is the result of a variety of biochemical reactions and microbial interactions which contribute to the organoleptic properties of wine. Wine aroma and flavour encapsulate the sensory experience of wine and could ultimately determine wine quality and consequently influence consumer acceptance and preference of a product. A thorough understanding of potential factors influencing wine aroma and flavour is therefore needed in order to exploit such factors. The aim of this study was to evaluate the influence of commercial malolactic fermentation (MLF) starter cultures on wine composition, aroma and flavour and the potential impact on consumer preference of experimentally produced red wines. An analytical platform was established to capture the compositional changes induced by different MLF bacteria in experimentally produced red wines. A fast gas chromatography flame ionisation detection (GC-FID) method was developed to determine 39 wine volatile compounds in less than 15 minutes per sample. A 3-fold reduction in analysis time was achieved in comparison to a conventional GC-FID method (40 minutes). Analytes quantified comprise a large boiling point and polarity range which illustrates the robustness of the method. A method was also developed for the direct quantification of carbonyl compounds including diacetyl, acetoin, 2,3-pentanedione and certain aldehydes using headspace solid phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME GC-MS). Both analytical methods showed satisfactory linearity, repeatability and limits of quantification. The contribution of four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures to the volatile composition, organic acid content and infrared spectral properties of experimentally produced South African red wines, showed significant strain-specific variations in the organic acid profiles, especially for the production of citric acid and lactic acid during MLF. Subsequently, concentrations of compounds related to citric acid metabolism, namely ethyl lactate, acetic acid, diacetyl and acetoin, were influenced accordingly. Bacterial metabolic activity increased the concentration of higher alcohols, fatty acids and esters, with a larger increase observed in ethyl esters compared to acetate esters. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, however, further investigation is needed to clarify this observation. Infrared spectral fingerprints were used to characterise the different bacteria and in addition, the prediction of MLF related compounds, diacetyl, acetoin and 2,3-pentanedione, from mid-infrared spectra was explored by partial least squares (PLS) models. Quantitative descriptive analysis (QDA) results depicted significant differences between wines fermented with different starter cultures, in terms of sensory attributes including buttery, fruity, nutty and yoghurt/buttermilk aroma as well as smoothness and mouth-feel attributes. Consumer preference testing results indicate that sensory differences imparted by different MLF bacteria could influence consumer-liking. Preference mapping revealed interesting relationships between sensory attributes and consumer-liking, that can be used for preliminary interpretative purposes. In conclusion, this study illustrated the potential impact of bacterial strains on wine aroma and flavour, resulting sensory properties and consumer preference through an integrative approach combining compositional, spectral, sensory and consumer data. The results presented in this study are of significance to the wine industry since they illustrate and reiterate the potential of different MLF starter cultures as an additional tool to contribute to wine aroma and flavour, and potentially influencing consumer preference and product liking. / AFRIKAANSE OPSOMMING: Wyn is die resultaat van ‘n verskeidenheid biochemiese reaksies en mikrobiologiese interaksies wat tot die organoleptiese eienskappe van die finale produk bydra. Wynaroma en geur vang die sensoriese ervaring van wyn vas en dit kan dus wynkwaliteit bepaal en gevolglik verbruikersaanvaarding asook voorkeur van ‘n produk beïnvloed. Die potensiële faktore wat wynaroma en geur kan beïnvloed moet dus vir hierdie rede deeglik bestudeer word ten einde sulke faktore ten volle te benut. Die doel van hierdie studie was om die invloed van kommersiële applemelksuurgisting (AMG) aanvangskulture op wynsamestelling, die gevolglike aroma en geur eienskappe en die potensiële impak op verbruikersvoorkeure te ondersoek. ‘n Analitiese platform is gevestig om die veranderings in samestelling veroorsaak deur verskillende AMG bakterieë in eksperimenteel bereide rooi wyne vas te vang. ‘n Vinnige gas chromatografiese vlam geïoniseerde deteksie (GC-FID) metode is ontwikkel vir die meting van 39 vlugtige komponente in minder as 15 minute per wynmonster. In vergelyking met ‘n konvensionele GC-FID metode (40 minute) is ’n 3-voudige vermindering in analise tyd behaal. Gekwantifiseerde komponente bestaan uit ‘n wye kookpunt- en polariteitsreeks wat die robustheid van die metode illustreer. ‘n Metode vir die direkte kwantifisering van karboniel komponente, insluitende diasetiel, asetoïen, 2,3-pentanedioon en verskeie aldehiede is ontwikkel met die gebruik van dampfase soliede fase mikroekstraksie gekoppel aan gas chromatografie massa spektrometrie (HS-SPME GC-MS). Albei analitiese metodes besit voldoende lineariteit, herhaalbaarheid en lae deteksie limiete. Die bydrae van vier kommersiële Oenococcus oeni AMG aanvangskulture tot die vlugtige samestelling, organiese suurinhoud en infrarooi spektrale eienskappe van Suid-Afrikaanse rooiwyn het beduidende ras spesifieke variasies in die organiese suur profiele, spesifiek vir die produksie van sitroensuur en melksuur gedurende AMG, vertoon. Gevolglik is die konsentrasies van komponente verwant aan sitroensuur metabolisme, naamlik etiellaktaat, asynsuur, diasetiel en asetoïen, dien ooreenkomstig beïnvloed. Bakteriese metaboliese aktiwiteit het ‘n toename tot gevolg gehad in die hoër alkohole, vetsure en algemene ester konsentrasies met ‘n groter toename in etiel-esters in vergelyking met asetaat-esters. ‘n Ras-spesifieke tendens om die totale aldehiedkonsentrasie te verminder na afloop van AMG, is waargeneem alhoewel verdere ondersoek in hierdie area nodig is. Infrarooi spektrale patrone is gebruik om verskillende bakterieë te karakteriseer asook om die voorspelling van spesifieke AMG verwante komponente soos diasetiel, asetoïen en 2,3-pentanedioon met die gebruik van mid-infrarooi spektrala parsiële kleinste kwadraat verskille (PLS) modelle te ondersoek. Kwantitiewe beskrywende sensoriese analise illustreer beduidende verskille tussen wyne wat gefermenteer is met verskillende aanvangskulture in terme van geure soos botteragtigheid, vrugtigheid, neutagtigheid, joghurt/karringmelkgeur, asook gladheid en mondgevoel eienskappe. Verbruikersvoorkeur resultate illustreer die groot invloed wat sensoriese verskille veroorsaak deur verskillende AMG bakterieë op verbruikersvoorkeure kan hê. Voorkeur kartering het interessante verhoudings tussen sensoriese eienskappe en verbruikersvoorkeure uitgelig. Hierdie studie illustreer die impak van bakteriese rasse op wynaroma en geur en verbruikersvoorkeure deur ‘n geïntegreerde benadering waarin samestellende, spektrale, sensoriese en verbruikersdata gekombineer is. Die resultate van hierdie studie is van belang vir die wynindustrie deurdat dit die potensiële bydrae van verskillende AMG kulture tot wynaroma en geur asook die potensiaal om verbruikersvoorkeure te beïnvloed, illustreer en beklemtoon.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: This study focused on the potential of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS) for the improved analysis of volatile wine constituents. Solid phase microextraction (SPME) in combination with GC×GC-TOF-MS was successfully used for the detailed investigation of the impact of three commercial Oenococcus oeni lactic acid bacteria (LAB) strains on the volatile composition of Pinotage wines subjected to malolactic fermentation (MLF). Due to increased separation power and enhanced sensitivity obtained by using two orthogonal separations coupled with the structural information provided by deconvoluted TOF-MS spectra, GC×GC-TOF-MS allowed for the identification and semi-quantitative analysis of much larger numbers of compounds compared to previous studies applying one-dimensional gas chromatography. The combination of univariate and multivariate statistical assessment was used as a powerful tool for data interpretation. The obtained results contribute significantly to the understanding of the impact of MLF on the volatile composition of Pinotage wine Some compounds have been linked to MLF for the first time. Moreover, the impact of these commercial starter cultures on the composition of volatile sulfur and nitrogen compounds in the same wines was studied by one-dimensional gas chromatographic methods with headspace injection and solid supported liquid-liquid extraction together with sulfur selective detection and tandem mass spectrometry. This study demonstrated also for the time, the impact of MLF on the composition of volatile sulfur and nitrogen compounds in Pinotage wine. GC×GC-TOF-MS was further used for the evaluation of the suitability of a new phase for stir bar sorptive extraction (SBSE) analysis of wine volatiles. Despite instrumental complications, beneficial extraction properties of the new stir bar phase for especially more polar compounds could be demonstrated. In addition, the extraction ability of this novel phase was evaluated for the analysis of selected thiazoles in wine using heart-cutting two dimensional gas chromatography in combination with nitrogen selective detection. Advantageous extraction performance of the new stir bar phase compared to a conventional polydimethylsiloxane (PDMS) phase for the determined thiazoles was demonstrated. / AFRIKAANSE OPSOMMING: Hierdie studie het gefokus daarop om die potensiaal van omvattende tweedimensionele gaschromatografie gekombineer met vlugtyd massaspektrometrie (GC×GC-TOF-MS) vir die verbeterde analise van vlugtige wynkomponente te ondersoek. Soliede fase mikro-ekstraksie (SPME) in kombinasie met GC×GC TOF MS is met sukses aangewend vir ‘n ondersoek na die impak van drie kommersiële Oenococcus oeni melksuur bakteria (LAB) rasse op die samestelling van die vlugtige fraksie van Pinotage wyne wat appelmelksuurgisting (AMG) ondergaan het. As gevolg van die verbeterde skeidingsvermoë en die verhoogte sensitiwiteit wat verkry word deur twee ortogonale skeidings te kombineer, tesame met die inligting aangaande die molekulêre struktuur wat die die gedekonvoleerde TOF massaspektra verskaf, maak GC×GC-TOF-MS die identifikasie en semi-kwantitatiewe analise van aansienlik meer komponente, in vergelyking met die gebruik van een-dimensionele gaschromatografie, moontlik. Die kombinasie van monoveranderlike asook multiveranderlike statistiese evaluering is gebruik as ‘n kragtige tegniek vir data interpretasie. Die resultate wat verkry is dra tot ‘n groot mate by tot die ontrafeling en begrip aangaande die impak wat AMG op die samestelling van vlugtige komponente in Pinotage wyn het. Daar word ook vir die eerste keer aangetoon dat somminge komponente verband te hou met AMG. Aanvullend hiertoe is die impak wat hierdie kommersiële kulture (wat gebruik word om fermentasie te inisieer) op die voorkoms van swawel en stikstof bevattende vlugtige komponente het bestudeer deur gebruik te maak van een-dimensionele gaschromatografiese metodes met ‘headspace’ inspuiting en vloeistof-voeistof ekstraksie tesame met swawel en stikstof selektiewe deteksie en tandem massaspektrometrie. Hierdie ondersoek werp lig, ook vir die eerste keer, op die samestelling van vlugtige swawel en stikstof bevattende komponente in Pinotage wyn. GC×GC-TOF-MS is ook gebruik vir die evalueering van die toepaslikheid van ‘n nuwe stasionêre fase vir gebruik met roerstaaf sorptiewe ekstraksie (SBSE) vir die analisering van vlugtige komponente in wyn. Ten spyte van instrumentele komplikasies, is die voordele wat hierdie nuwe fase vir die ekstraksie van vernaamlik meer polêre komponete aangetoon. Vervolgens is die ekstraksievermoë van hierdie nuwe fase vir die analise van sekere tiasole in wyn met ‘heart-cutting’ twedimensionaly gaschromatografie in kombinasie met stikstof-selektiewe deteksie gedemonstreer. Verbeterde ekstraksie van die nuwe roerstaaf fase vir die analise van tiasole, in vergelyking met ‘n tradisionele polydimethylsiloxane (PDMS) fase is voorts aangetoon.

Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The production of good quality wine is essential to ensure competitiveness on an international level. Wine quality is usually evaluated for the visual, olfactory and taste characteristics of that specific wine. The winemaking process starts with the grapes in the vineyard followed by oenological practises in the winery until the final wine is bottled. Factors that could influence wine quality include the grape quality from which the wine is made and different techniques used during wine production. Other factors include the presence as well as the interaction between microorganisms found in the grape juice and wine, and the biochemical effect these microorganisms have on certain chemical compounds in the wine. The different microorganisms found in grape juice and wine can either have a negative or positive contribution to the final quality of the wine. During certain stages of the winemaking process the growth and metabolic activity of certain microorganisms is a necessity to produce good wine. During other stages the presence of certain microorganisms can lead to the development of compounds that is regarded as off-flavours and therefore lead to unpalatable wines of low quality. Yeast strains that naturally present on the grapes and in the winery can also contribute to the final quality of the wine. Brettanomyces yeasts are part of the natural flora of winemaking and can drastically influence the aroma characters of a wine through the production of volatile phenols. The general aroma descriptions of volatile phenols include "smoky", "spicy", "barnyard", "animal" and "medicinal". Although some wine drinkers believe that these characters can add to the complexity of a wine, high levels of volatile phenols is mostly regarded as off-flavours and mask the natural fruity flavours of a wine. With this study we wanted to generate a better understanding of the effect of different winemaking practises on the production of volatile phenols by B. bruxellensis. We evaluated the difference in volatile phenol production when B. bruxellensis was introduced before or after alcoholic fermentation. We have shown that B. bruxellensis could grow and produce volatile phenols during alcoholic fermentation. Results obtained also showed that commercial wine yeast strains could produce the vinyl derivatives that serve as precursors for Brettanomyces yeast to produce the ethyl derivatives. The commercial yeast strains differed in their ability to produce vinyl derivatives. Different malolactic fermentation scenarios were evaluated, namely spontaneous versus inoculated, and with or without yeast lees. Results showed that spontaneous malolactic fermentation had higher volatile phenol levels in the wine than inoculated malolactic fermentation. The treatment with lees reduced the level of volatile phenols, probably due to absorption by yeast cells. The presence of the phenyl acrylic decarboxylase (PAD1) gene and the production of volatile phenols by S. cerevisiae commercial yeast strains were evaluated in Shiraz grape juice and in synthetic grape juice. The results indicated that the yeast strains differ in their ability to produce 4-vinylphenol and 4-vinylguaiacol. All the yeast strains tested had the PAD1 gene. We also evaluated the presence of the phenolic acid decarboxylase (padA) gene and the ability of different lactic acid bacteria strains to produce volatile phenols in synthetic wine media. Although some of these strains tested positive for the phenolic acid decarboxylase gene most of them only produced very low levels of volatile phenols. This study made a valuable contribution on the knowledge about the effect of Brettanomyces yeast on the volatile phenol content of red wines during different stages of the winemaking process and when applying different winemaking practices. It also showed the effect between Brettanomyces yeast and other wine microorganisms and the possible influence it could have on the final quality of wine. Research such as this can therefore aid the winemaker in making certain decisions when trying to manage Brettanomyces yeast spoilage of wines.

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.

Oenococcus oeni est la principale espèce de bactérie lactique d’intérêt oenologique, elle réalise la plupart des fermentations malolactiques. Les souches d’O. oeni ont souvent été étudiées dans le but de sélectionner des levains malolactiques. Ces souches possèdent des caractéristiques génétiques, et phénotypiques variées. Néanmoins, l’utilisation de différentes méthodes de typage n’a pas permis à ce jour d’obtenir une image exhaustive de la diversité oenologique de cette espèce. L’intérêt grandissant des viticulteurs pour mieux maitriser les FML spontanées nécessite de mieux connaitre cette diversité.La population de souches indigènes d’O. oeni présentes dans les FML de différentes régions a été étudiée, pour tenter d’obtenir une image plus complète de la diversité de cette espèce et savoir s’il serait pertinent de sélectionner des souches de régions ou d’exploitations. L’analyse d’un très grand nombre de vins a mis en évidence la présence de souches spécifiques aux régions, appellations, produits et exploitations étudiées. Un génotypage à l’aide de SNP a permis de les classer dans les groupes génétiques déjà connu et a confirmé l’existence de groupes spécifiques à un type de produit. Certaines de ces groupes rassemblent des souches présentant des comportements phénotypiques et des capacités fermentaires similaires. En complément de l’analyse de diversité, des essais ont été menés pour proposer des protocoles de production de souches et d’utilisation de lies pour améliorer la réalisation des FML spontanées. L’ensemble de ces travaux soulignent la grande diversité génétique de l’espèce O. oeni, l’étude des souches spécifiques de type de vin pourrait aider à comprendre son adaptation à cet environnement. / Oenococcus oeni is the main species of lactic acid bacteria of oenological interest, it performs most of malolactic fermentation. O. oeni strains have often been studied to select malolactic starters. These strains have various genetic and phenotypic characteristics. However, the use of different typing methods failed so far to obtain a complete picture of the enological diversity of this species. The growing interest of winegrowers to better control the spontaneous requires a better knowledge of this diversity.The population of indigenous O. oeni strains present during MLF in different regions has been studied in an attempt to obtain a more complete picture of the diversity of the species and to know whether it would be appropriate to select strains of regions or wineries. The analysis of a large number of wines showed the presence of strains specific to wineproducing areas, different kind of wines, and wineries. SNP Genotyping allowed us to classify these strains in the already known genetic groups and confirmed the existence of specific groups to a specific kind of product. Some of these strains belonging to the same specific genetic groups showed similar behavior and technological properties. In addition to the diversity analysis, trials of production of strains and use of lees to ameliorate the MLF have been done to improve the achievement of spontaneous MLF. Taken together, these studies emphasize the high genetic diversity of the species O. oeni, the study of specific strains of type of wine could help understand the adaptation of this species to this environment.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Hen egg white lysozyme (HEWL; muramidase; EC 3:2:1:17) is an enzyme present in high concentrations in chicken (Gallus gallus) egg whites. It hydrolyses the link between N-acetylmuramic acid and N-acetylglucosamine in Gram positive bacterial cell walls, resulting in cell death. It is thus active against lactic acid bacteria (LAB), which may be present in grape juices and musts. These bacteria are responsible for malolactic fermentation of wines although many species, particularly of the genera Lactobacillus and Pediococcus, are considered spoilage organisms. The growth of LAB is therefore closely monitored and controlled during winemaking. The most common means of control is growth inhibition by chemical treatment (usually with SO2). Lysozyme is a commonly used wine processing aid, complementing the antimicrobial activity of SO2 . It allows for lower doses of SO2 to be used, thus improving the wholesomeness of wine. The OIV (Organisation Internationale de la Vigne et du Vin) approved its use in quantities up to 500 mg per liter of wine in 1997. This study evaluated the effect of different secretion signals on the secretion of lysozyme by the haploid auxotroph Saccharomyces cerevisiae strain FY23. Secretion by an industrial strain (VIN13) transformed with a single copy of the HEWL gene with the MF-a secretion signal under the control of the PGK1 (phosphoglycerate kinase 1) prompter and terminator was also evaluated. In the case of FY23 four secretion signals were used, namely the native lysozyme signal and the S. cerevisiae mating factor-a signal as well as mutants of these signals. These mutants incorporated two additional arginines at the N-terminus of the signals immediately downstream of the terminal methionine. The effect of these mutations was to increase the positive charge of the secretion signal N-terminals. The secretion signal-lysozyme fusions were placed under the regulation of the S. cerevisae PGK1 gene’s promoter and terminator. The resulting expression cassettes were cloned into integrating vectors YIpLac211 and pDMPOF1b and episomal vector pHVX2. These were used to transform FY23 and VIN13. FY23 as well as VIN13 transformants were evaluated in an artificial medium designed to reflect the nutrient content of grape juice, with particular attention being paid to assiminable nitrogen. Three hexose concentrations were tested in order to determine the effect thereof on lysozyme secretion titer. Lysozyme secreted under all tested growth conditions was found to be too low for detection by either enzymatic assay or HPLC-FLD. For this reason secreted lysozyme was isolated and concentrated 10x by means of cation-exchange. Subsequently, lysozyme concentrations in the concentrates was determined by means of the aforementioned techniques. SDS-PAGE analysis of lysozyme concentrates was also performed. No significant differences were found between native or MF-a secretion signals and their mutated counterparts in terms of secretion titer or proteolytic maturation. Lysozyme secreted with the MF-a signal was found to be misprocessed in all cases, with both an authentically processed and a larger form, in which the secretion signal was not completely removed, being present. Lysozyme secreted with the native signal appeared to be correctly processed in all cases. Secretion titer from high copy number episomal FY23 tranformants was similar to that of integrants containing a single copy of the gene. Sugar concentration affected lysozyme production, with higher quantities of the enzyme being secreted when higher initial sugar concentrations were used. Lysozyme titers were extremely low (< 0:25 mg/L) with all expression cassettes under all the tested conditions with both FY23 and VIN13. In the case of the VIN13’s a lower final biomass was found for the secretor strain tested in comparrison to the VIN13 wild-type. / AFRIKAANSE OPSOMMING: Hoendereierwitlisosiem (HEWL; muramidase, EG 3:2:1:17) is ´n ensiem teenwoordig in hoë konsentrasies in hoender (Gallus gallus) eierwitte. Dit hidroliseer die binding tussen N-asetielmuramiensuur en N-asetielglukosamien in Gram positiewe bakteriese selwande, wat tot seldood lei. Dit is dus aktief teen melksuurbakterieë (MSB), wat in druiwesap en mos teenwoordig kan wees. Hierdie bakterieë is verantwoordelik vir appelmelksuurgisting van wyne, hoewel baie spesies, veral van die genera Lactobacillus en Pediococcus, ook as bederforganismes beskou word. Die groei van MSB word dus noukeurig tydens wynbereiding gemoniteer en beheer. Die algemeenste wyse van beheer is groei-inhibisie deur chemiese behandeling (gewoonlik SO2). Lisosiem is ´n algemeen gebruikte wyntoevoegingsmiddel en vul die antimikrobiese aktiwiteit van SO2 aan. Met lisosiem kan ´n laer dosis van SO2 gebruik word, wat lei tot ´n verbetering van die heilsaamheid van die wyn. Die OIV (Organisasie Internationale de la Vigne et du Vin) het die gebruik daarvan goedgekeur tot en met 500 mg per liter wyn vanaf 1997. Hierdie studie het die effek van verskillende sekresieseine op die uitskeiding van lisosiem deur die haploïede ouksotrofe Saccharomyces cerevisiae stam, FY23, geëvalueer. Uitskeiding deur ´n industriële stam (VIN13), wat getransformeer is met ´n enkelkopie van die HEWL-gene met die MF-a sekresiesein onder die beheer van die PGK1 (Fosfogliseraat kinase 1) promotor en termineerder, is ook geëvalueer. In die geval van FY23 is vier sekresieseine gebruik, naamlik die inheemse lisosiemsein, S. cerevisiae MF- a sein, asook mutante van hierdie seine. Hierdie mutante het twee bykomende arginienresidu’s by die N-terminus van die seine direk stroom-af van die terminale metionien. Die effek van hierdie mutasies was om die positiewe lading van die uitskeidingsein N-terminale te verhoog. Die gevolglike uitdrukkingskassette is in die integrasievektor YIpLac211 en pDMPOF1b, en die episomale vektor pHVX2, gekloneer. Dit is gebruik om VIN13 en FY23 te transformeer. FY23, sowel as VIN13-transformante, is geëvalueer in ´n kunsmatige medium wat ontwerp is om die voedingsinhoud van druiwesap te weerspieël, met besondere aandag aan assimileerbare stikstof. Drie heksose konsentrasies is getoets om te bepaal wat die uitwerking daarvan op die lisosiemsekresietiter is. Onder alle groeitoestande was die isosiem wat uitgeskei is, te laag om deur ensimatiese toetse of HPLC-FLD bepaal te word. Om hierdie rede is uitgeskeide lisosiem geïsoleer en 10x gekonsentreer deur middel van katioon-uitruiling. Daarna is lisosiemkonsentrasies bepaal deur middel van bogenoemde tegnieke. SDS-PAGE-ontleding van lisosiemkonsentraat is ook uitgevoer. In terme van sekresietiter of proteolitiese maturasie, is geen beduidende verskille gevind tussen inheemse of MF-a sekresieseine en hul gemuteerde eweknieë nie. Lisosiem wat deur die MF-a sein uitgeskei is, is in alle gevalle foutief geprosesseer, met ´n teenwoordigheid van beide die regte produk en ´n groter produk, waarin die uitskeidingsein nie heeltemal verwyder word nie. Lisosiem wat met die inheemse sein uitgeskei is, blyk in alle gevalle korrek verwerk te wees. Sekresietiter van ´n aantal hoë-kopie episomale FY23-transformante was soortgelyk aan dié van integrante met ´n enkelkopie van die geen. Suikerkonsentrasie beïnvloed lisosiemproduksie, met ´n hoër hoeveelheid van die ensiem wat uitgeskei word wanneer die aanvanklike suiker in hoër konsentrasies gebruik is. Lisosiemtiters was baie laag (< 0:25 mg/L), met al die kassette onder al die getoetste toestande vir beide FY23 en VIN13. In die geval van die VIN13’s, is ´n laer finale biomassa vir die uitskeidingstam in vergelyking met die VIN13 wilde-tipe gevind.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The influence of malolactic fermentation (MLF) in most red and some white wines is one of many factors that determine or influence wine quality, because it affects the flavour and sensory profile of wine. This process is a decarboxylation process conducted by lactic acid bacteria (LAB) such as Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Mostly Oenococcus oeni, but recently also Lactobacillus plantarum is used in commercial starter cultures and also the first mixed MLF starter culture (NT 202 Co-Inoculant) was commercialized in 2011. The reason for the predominant use of O. oeni and recently L. plantarum is due to their tolerance to the harsh wine environment. Malolactic fermentation leads to a decrease in acidity and an increase in pH that leaves the wine with a softer mouthfeel. Another reason to conduct MLF is the improvement of microbial stability by the removal of malic acid as carbon source. Research focus has recently shifted to the ability of LAB and MLF as well as the interaction of LAB with yeast to alter the wine aroma profile via the modification and/or production of certain aroma compounds. The main goal of this study was to assess the impact of yeast and nutrient addition on the ability of the NT 202 Co-Inoculant to conduct MLF during co-inoculation and to evaluate the aroma compound production in the final wine. The first aim was to evaluate the impact of different red and white wine yeast strains on the ability of the NT 202 Co-Inoculant to conduct MLF during co-inoculation in Chardonnay, Merlot and Shiraz. Malolactic fermentation was unsuccessful in the Chardonnay due to a low pH, but successful in Merlot and Shiraz. Based on the malic acid degradation ability of the NT 202 Co- Inoculant, the yeasts were grouped into three categories: inhibitory, neutral or stimulatory towards MLF. Co-inoculated MLF showed a clear decrease in total fermentation time while yeast strains such as WE 372 and Exotics showed positive compatibility with the NT 202 Co- Inoculant. The impact of the yeast-bacterial combinations on the aroma compound production in the final wine was evaluated. Co-inoculated MLF showed positive aroma changes in the red wines with a general increase in total esters (associated with fruity characters in wine) especially ethyl lactate and diethyl succinate that also contribute to the mouthfeel of the wine. Production of esters, volatile fatty acids and higher alcohols seemed to depend on the yeastand LAB strain used. The NT 202 Co-Inoculant contributed to the monoterpenes produced and MLF led to increased concentrations of diacetyl and acetoin, which are associated with buttery characters in wine. The second aim of this study was to evaluate the impact of wine additives (used during coinoculation) such as yeast- and bacterial nutrients, clarifying- and detoxifying agents on the ability of the NT 202 Co-Inoculant to conduct MLF and to assess their impact on the aroma compound production in the final wine. No negative or positive impact on the malic acid degradation of the NT 202 Co-Inoculant or the resulting aroma compound production was observed for the different wine additives used in this study. The results generated from this study showed that the selection of yeast strains is important as it will influence both the fermentation duration and final wine aroma. / AFRIKAANSE OPSOMMING: Die invloed van appelmelksuurgisting (AMG) in die meeste rooi- en witwyne is een van baie faktore wat wynkwaliteit beïnvloed, omrede dit die geur en sensoriese profiel van wyn beïnvloed. Hierdie proses is 'n dekarboksileringsaksie wat deur melksuurbakterieë (MSB), soos Oenococcus, Lactobacillus, Pediococcus en Leuconostoc, uitgevoer word. Die mees algemene bakterieë wat gebruik word, is Oenococcus oeni, maar onlangs het Lactobacillus plantarum ook na vore getree in die gebruik van kommersiële aanvangskulture. Die eerste gemengde AMGaanvangskultuur (NT 202 Co-Inoculant) is in 2011 gekommersialiseer. Die rede vir die oorheersende gebruik van O. oeni en L. plantarum word toegeskryf aan hul gehardhiedsgraad in ‘n uitdagende wynomgewing. Appelmelksuurgisting lei tot 'n afname in die suurheidsgraad en 'n toename in die pH van die wyn, wat 'n sagter mondgevoel tot gevolg het. Nog 'n rede waarom AMG deurgevoer word, is om die mikrobiese stabiliteit van die wyn te verbeter deur die verwydering van appelsuur as koolstofbron. Die navorsingsfokus het onlangs verskuif na die vermoë van MSB en AMG, sowel as die interaksie van MSB met die gis, om die wynaromaprofiel te verander deur middel van die verandering en/of produksie van sekere aromaverbindings. Die hoofdoel van hierdie studie was om die impak van die gis en voedingstof te evalueer ten opsigte van die vermoë van die NT 202 Co-Inoculant om AMG uit te voer tydens koïnokulasie. Die produksie van aromakomponente in die finale wyn is ook geëvalueer. Die eerste doelwit was om die impak van verskillende rooi- en witwyngisrasse te evalueer ten opsigte van die vermoë van die NT 202 Co-Inoculant om AMG uit te voer tydens koïnokulasie in Chardonnay, Merlot en Shiraz. Appelmelksuurgisting was onsuksesvol in die Chardonnay weens 'n lae pH, maar suksesvol in Merlot en Shiraz. In terme van die appelsuurafbraakvermoë van die NT 202 Co-Inoculant, is die giste in drie kategorieë gegroepeer: inhiberend, neutraal of stimulerend teenoor AMG. Ge-koïnokuleerde AMG het 'n duidelike afname in die totale fermentasietyd getoon, terwyl gisrasse, soos WE 372 en Exotics, ‘n positiewe verenigbaarheid met die NT 202 Co-Inoculant getoon het. Die impak van die gisbakteriële kombinasies op die aromakomponentproduksie in die finale wyn is geëvalueer. Gekoïnokuleerde AMG het positiewe aromaveranderinge in die rooiwyne getoon met 'n algemene toename in die totale esters (wat geassosieer word met vrugtige karakters in wyn), veral etiellaktaat en dietielsuksinaat, wat ook bydra tot die mondgevoel van die wyn. Dit het voorgekom dat produksie van esters, vlugtige vetsure en hoër alkohole moontlik afhanklik kan wees van die gis- en bakteriële ras gebruik. Die NT 202 Co-Inoculant het bygedra tot die monoterpene wat geproduseer is en AMG het gelei tot verhoogde konsentrasies van diasetiel en asetoïen, wat geassosieer word met botteragtige karakters in wyn. Die tweede doelwit van hierdie studie was om die impak van wyntoevoegingsmiddels (wat tydens koïnokulasie gebruik word) bv. gis- en bakteriese voedingstowwe, verhelderingsagente, asook detoksifiserende agente, te evalueer ten opsigte van die vermoë van die NT 202 Co- Inoculant om AMG uit te voer en om hul impak op die produksie van die aromakomponente van die finale wyn te ontleed. Geen negatiewe of positiewe effekte is waargeneem vir die verskillende wyntoevoegingsmiddels, wat in hierdie studie gebruik is, in terme van die appelsuurafbraak van die NT 202 Co-Inoculant of die gevolglike produksie van aromakomponente nie. Hierdie studie se resultate toon dat die keuse van die gisras belangrik is, omdat dit die fermentasietydperk, asook die finale wynaroma, beïnvloed. / Anchor Yeast, Oenobrands, The National Research Foundation and THRIP, for financial support

Thesis (MSc)--Stellenbosch University, 2007. / ENGLISH ABSTRACT: With ever increasing pressure on wine producers to lower the financial costs involved in winemaking to be able to compete in the market, all while maintaining a high level of wine quality, the focus on maintaining control over all aspects of the winemaking process are greatly emphasized. Malolactic fermentation (MLF) is one of the important processes in red wine production. The advantages of this process, when performed successfully, is widely known and accepted. One way to gain control over MLF is the use of MLF starter cultures. Starter cultures usually consist of Oenococcus oeni that has been isolated from grapes or wines and is in most cases available in a freeze-dried form ready for direct inoculation into the wine when MLF is desired. Starter cultures are induced into wine and usually ensure the immediate onset as well as a fast and clean execution of the process. Starter cultures used in South Africa are in most cases isolated from cooler viticultural regions in the Northern hemisphere. The constitution of wines from cooler viticultural regions, differ from those in South Africa, which has a warm climate. The most important difference is the acid content of the wines which is lower in South African must/wines and results into a higher pH. The three most important changes that develop in wine during MLF are a decrease in acidity due to the conversion of malic acid to the less harsh lactic acid, enhanced flavour and aroma of wine and an increase in the microbiological stability of wine. The decrease in acidity is very important for wines produced for grapes grown in cool viticulture regions. In South Africa though, the climate is warm and higher pH’s are present in the musts and wines and the de-acidification due to MLF is not the main aim but rather the microbiological stabilisation. One of the compounds that could be produced by lactic acid bacteria (LAB) is biogenic amines (BA’s). These compounds can be hazardous to human health. This thesis focussed on the performance of MLF starter cultures in high pH South African red wines. The first objective of the study was to stretch MLF starter cultures in high pH red wines of South Africa. Stretching means to use less than the prescribed dosage or the re-use of starter cultures. The difference in MLF rate, the influence of the natural occurring LAB and the levels of biogenic amines formed during MLF were determined for the different stretching treatments. The results showed that different rates in malic acid degradation were experienced between the treatments, but in all cases MLF fermentation was completed. Biogenic amines were formed at various levels and the influence of the natural occurring LAB also played a role. The second objective of the study was the evaluation of the effect of a wine isolated LAB (Lactobacillus) and an acetic acid bacteria (AAB), inoculated with a MLF starter culture had on MLF at different wine pH’s. It was found that especially in the case where the Lactobacillus was inoculated in combination with the MLF starter culture a possible stimulatory effect was experienced with regards to malic acid degradation rate. Biogenic amine concentration was measured at the end of MLF and it was found that no histamine and tyramine were formed in any of the treatments, while the putrescine and cadaverine levels were found to be at approximately similar levels for the different treatments. The third objective was to evaluate the possible influence of commercial tannin additions and a pectolytic enzyme on rate of MLF and phenolic composition of high pH red wine. The commercial tannins had possible inhibitory as well as stimulatory effects on the rate of malic acid degradation especially during the initial stages of MLF, with the highest dosage having the significant effect. The BA results showed difference in the levels produced due to tannin additions as well as strain differences could exist. The phenolic content showed a decrease in colour density, total red pigments, total phenolics and anthocyanins between AF and MLF. The fourth objective was to evaluate inoculation time of MLF starter cultures. The results showed that the fastest AF/MLF time was with simultaneous inoculation of the yeast and MLF starter cultures. It was also for this treatment where no histamine or tyramine was detected at the end of MLF compared to the other inoculation strategies (before the end of AF and after AF). This study generated a large amount of novel data which made a valuable contribution with regards to MLF in high pH red wines of South Africa. / AFRIKAANSE OPSOMMING: Die druk om wyne van hoë gehalte teen lae insetkoste te lewer om deel te bly van ’n kompeterende mark, plaas die fokus weer sterk op onder andere die beheer van alle aspekte van die wynmaak proses. Appelmelksuurgisting (AMG) is een van die belangrikste prosesse van rooiwyn produksie. Die voordele van AMG, in die geval van die suksesvolle implementering daarvan is vandag bekend en word geredelik aanvaar. Een van die metodes om beheer te verkry oor the proses van AMG is deur die gebruik van AMG aanvangskulture. AMG aanvangskulture bestaan uit Oenococcus oeni wat geïsoleer word vanaf druiwe of mos/wyn en is in meeste gevalle beskikbaar in ’n gevries-droogte vorm wat direk in wyn geïnokuleer kan word. Aanvangskulture word in wyn geïnduseer om die onverpose aanvang van AMG te bewerkstellig asook om ’n vinnige en skoon deurvoering van die proses te verseker. Die aanvangskulture wat in Suid-Afrika vir hierdie doeleinde gebruik word is in meeste van die gevalle verkry uit koue wingerdbou gebiede in die Noordelike Halfrond. Die samestelling van druiwe van koue wingerdbou gebiede en dié van Suid-Afrikaanse warm wingerdbou gebiede verskil. Die belangrikste verskil word ervaar in die suur inhoud, wat laer is in Suid-Afrikaanse druiwe en dus lei tot ‘n hoër pH inhoud. Die drie mees belangrikste veranderinge wat gedurende AMG in wyn plaasvind is die vermindering van die suur, as gevolg van die omskakeling van appelsuur na melksuur, die verbetering van die aroma en geur van wyn en die verbeterde mikrobiologiese stabiliteit. Die afname in suur is veral belangrik in wyne van koue wingerbou gebiede omdat die suur-inhoud daarvan soveel hoër is. In Suid-Afrika kan hierdie verlaging in suur egter lei tot ’n verdere verhoging in die pH wat plat wyne en uiteindelik ’n verlaging in die kwaliteit van wyn tot gevolg kan hê. Biogene amiene (BA) is verbinding wat melksuurbakterieë (MSB) kan vorm gedurende AMG en kan ernstige implikasies hê vir die mens se gesondheid. Hierdie tesis fokus op die evaluering van AMG aanvangskulture in hoë pH rooi wyne van Suid-Afrika. Die eerste doelwit gedurende hierdie studie was om AMG kulture te rek en die invloed daarvan in hoë pH rooiwyn te evalueer ten opsigte van the tempo van AMG, die rol van die natuurlike MSB te bestudeer asook om die vlak van biogene amiene te bepaal vir die verskillende behandelings. Die resultate het aan die lig gebring dat die rek van kulture verskille in die tempo van appelsuur afbraak tot gevolg het, maar dat AMG in alle gevalle wel suksesvol deurgevoer kon word. Die BA’e wat gevorm is, was teenwoordig in verskillende hoeveelhede. Die tweede doelwit was om die effekt van die gesamentlike inokulasie van ’n wyn geisoleerde MSB (Lactobacillus) asook ’n asynsuurbakterie (ASB) met ’n kommersiële AMG aanvangskultuur op AMG te evalueer. Hierdie eksperiment is uitgevoer by verskillende pH’s. Daar is gevind dat veral in die kombinasie inokulasie met die Lactobacillus, die tempo van appelsuur afbraak moontlik gestimuleer was. Geen histamien of tiramien is tydens AMG gevorm in hierdie eksperiment gevorm nie, terwyl putresien en kadaverien teenwoordig was teen ongeveer gelyke vlakke vir die behandelings. Die derde doelwit was om die moontlike invloed van kommersiële tannien toevoegings en die toevoeging van ’n pektolitiese ensiem te evalueer ten opsigte van AMG tempo die fenoliese samestelling van rooiwyn te bestudeer. Verskillende kommersiële tanniene het ’n moontlike sowel as inhiberende uitwerking gehad, veral gedurende die aanvanklike stadium AMG. Die grootste verskille is waargeneem in die behandelings waar die hoogste dosisse tannien bygevoeg is. Die BA resultate toon dat verkillende vlakke geproduseer was en dat hierdie verskille onstaan het as gevolg van verskille in tannien dosisse sowel as aanvangskulture. Die fenoliese inhoud het ’n afname in kleur intensiteit, totale rooi pigmente, totale fenole en antosianiene getoon vir die periode vanaf AF tot die einde van AMG. Die vierde doelwit was om the tyd van inokulasie van AMG aanvangskulture te bestudeer. Die resultate het getoon dat die vinningste tydperk van AF/AMG was ondervind in die geval waar die gis aanvangskulture gelyktydig met die AMG aanvangskulture geïnokuleer was. Geen histamine en tyramine het ook in hierdie behandeling ontwikkel nie, terwyl daar wel vlakke teenwoordig was in die ander behandelings (inokulasie net voor die einde van AF en na afloop van AF). Tydens hierdie studie is ’n groot hoeveelheid nuwe data geskep wat ‘n groot bydrae ten opsigte van AMG in hoë pH rooi wyne vanaf Suid-Afrika kan lewer.