Thesis (MSc)--Stellenbosch University, 1964. / ENGLISH ABSTRACT: The influence of manganese sulphate sprays on the yield and ripening of fruit of manganese-deficient Vitis vinifera L. (cv. Pinotage) plants was investigated. Ripening was determined in terms of changes in concentration of the indivi= dual and total sugars and organic acids. Increasing concentrations of manganese sulphate resulted in significant increases in the manganese content of the leaves. The higher levels of manganese were associated with an increase in yield. Ripening, however, was retarded, in that the percentage sugar of the fruit was decreased and titrable acid content increased. The principal sugars present in the fruit were sucrose, glucose, fructose and xylose. Malic and tartaric acids were the main organic acid constituents. Glucose and fructose increased sharply, and sucrose and xylose slightly during ripening of the fruit, whereas malic and tartaric acid de= creased. Glucose, fructose, tartaric and malic acid tended to increase with increasing manganese content. Sucrose and xylose were not appreciably affected. An application of 1% manganese sulphate can be recommend- · ed for manganese deficient vineyards, as it results in an increased yield, in addition to delaying ripening until a more favourable time for handling. Furthermore, the lowered sugar content of the fruit may be advantageous for the pro= duction of dry wines from Pinotage grapes . / AFRIKAANSE OPSOMMING: Die invloed van mangaansulfaatbespuiting op die opbrengs en vrugrypwording van Vitis vinifera L. cv. Pinotage, wat aan ernstige mangaantekorte gely het, is ondersoek. Rypwording is met betrekking tot veranderings in die konsentrasies van totale en indiwiduele suikers en sure bepaal. 2. Toenemende konsentrasies Mnso4 (van O.2% tot l.O%) het n betekenisvolle toename in die mangaangehalte van die blare (van 3 tot 80 d.p.m.) tot gevolg gehad. Die ver= hoogde mangaangehalte het gepaard gegaan met n toe= name in opbrengs, terwyl rypwording vertraag is, deurdat die suikerpersentasie en die titreerbare suurgehalte ver= meerder is in vergelyking met die kontroleplante. 3. Die vernaamste suikers in die vrugte was sukrose, glukose, fruktose en xilose. Glukose en fruktose het vinnig en sukrose en xilose geleidelik toegeneem met die verloop van rypwording. In die ryp vrugte was glukose en fruk= tose oorheersend. Glukose en fruktose was geneig om toe te neem met toenemende mangaangehalte, terwyl sukrose en xilose nie beinvloed is nie. 4. Appelsuur en Wynsteensuur was die oorwegende sure in die vrugte. Beide hierdie sure het gedurende rypwording verminder. Hulle konsentrasies het oor die algemeen toegeneem namate die mangaankonsentrasie verhoog is. 5. Dit is afgelei dat bespuiting met 1.0% MnS04 aanbeveel kan word vir wingerde met mangaantekorte, daar dit n toename in opbrengs, gepaard met 'n vertraging in rypwording tot gevolg gehad het. So 'n vertraging mag moontlik in parstyd voordelig wees. Die verlaagde suikerpersentasie van die vrugte is moontlik gunstig vir die bereiding van droe wyne, waarvoor Pinotage hoofsaaklik gebruik word.

Thesis (MScAgric)--Stellenbosch University, 1974. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Phenolic compounds are important quality indicators of a red wine, as they can contribute to the colour of a young red wine, colour stability during ageing as well as astringency, bitterness, body and overall mouthfeel properties. Wine composition is commonly influenced by winemaking and viticultural practices. In South Africa it often happens that vines are excessively vigorous, resulting in canopies that are too dense, which in turn could have a negative effect on the quantity and quality of the grapes produced. Viticultural practices such as judicious canopy management and irrigation are designed to control vine vigour and yield, thus improving fruit ripening and colour development. Artificial shading and water deficit have been reported to have an influence on the sensory properties of red wine as well as on the flavonoid composition. These effects are dependent on a number of factors, however, including the season, cultivar, light intensity, and the extent and timing of water deficit. There is limited research on the possible interactive effects of grapevine water deficits and canopy manipulation on grape and wine flavonoid composition in Shiraz, as well as the relationships between berry and wine composition. We thus investigated the effect of canopy reduction in combination with water deficit on the phenolic and colour composition of Shiraz grapes at different levels of ripeness, and in their corresponding wines after alcoholic and malolactic fermentation as well as after six months’ ageing. This study found that it is possible to improve the phenolic composition of grapes and wine by shoot removal, and some of the tendencies in the wines were also observed after the ageing period. If the shoot removal is not performed at a very early stage, sunburn damage can occur and this will result in berries with a lower mass and volume at harvest due to excessive exposure without the berry having adapted to the imposed conditions. Harvesting at different ripeness levels also affected the chemical and phenolic composition of the grapes and resulting wines. The water deficit effect on most phenolic parameters measured in the grapes and wine was not as prominent as that of the canopy manipulation treatment. This study improved our understanding of how an improvement in the canopy microclimate of Shiraz could be reflected in the phenolic composition of wines, along with a potentially important effect of harvesting date. On this basis it may be possible to attain a specific wine style. Harvesting at a ripe stage, for example, could result in the production of wines with higher colour density and astringency, while unripe grapes could result in wines with higher levels of perceivable fresh berry attributes. In particular, canopy reduction could increase the astringency and body of wines made from grapes subjected to water deficit. / AFRIKAANSE OPSOMMING: Fenoliese verbindings is belangrike kwaliteitsparameters van rooiwyn, aangesien dit kan bydra tot die kleur van ‘n rooiwyn, kleurstabiliteit tydens veroudering sowel as frankheid, bitterigheid en mondgevoel. ‘n Wyn se fenoliese samestelling word algemeen bepaal deur wynmaak- en wingerdkundige praktyke. In Suid-Afrika gebeur dit gereeld dat wingerde uitermatig groeikragtig is, wat lei tot te digte lower wat sodoende ‘n negatiewe effek op kwantiteit en kwaliteit van die druiwe wat geproduseer word, het. Wingerdkundige praktyke soos oordeelkundige lowerbestuur en besproeiing is ontwerp om wingerdstokke se groeikrag en opbrengs te beheer, en sodoende vrugrypwording en kleurontwikkeling te verbeter. Kunsmatige beskaduwing en waterstres is gerapporteer om ‘n invloed te hê op die sensoriese eienskappe van rooiwyn sowel as op die flavonoïedsamestelling. Hierdie effekte is egter afhanklik van ‘n verskeidenheid faktore, insluitende die seisoen, kultivar, ligintensiteit en die mate en tyd van waterstres toegepas. Daar is beperkte navorsing op die moontlike interaktiewe effekte van waterstres en lowermanipulasie op die druif en wyn flavonoïedsamestelling in Shiraz, sowel as die verhoudings tussen druif en wyn samestelling. Ons het dus die effekte van lowerbestuur in kombinasie met waterstres op die fenoliese en kleursamestelling van Shiraz druiwe by verskillende rypheidsvlakke ondersoek, asook in hul ooreenstemmende wyne na alkoholieseen appelmelksuurfermentasie sowel as na ses maande veroudering. Hierdie studie het gevind dat dit moontlik is om die fenoliese samestelling van druiwe en wyn deur lootverwydering te verbeter, en sommige van die tendense is ook waargeneem in die wyn na die verouderingsperiode. Indien lootverwydering nie toegepas word by ‘n baie vroeë stadium nie, kan sonbrand voorkom en dit kan lei tot korrels met ‘n laer massa en volume by oes as gevolg van oormatige blootstelling sonder dat die korrel aangepas het by die spesifieke kondisies. Oes by verskillende rypheidsvlakke affekteer ook die chemiese en fenoliese samestelling van die druiwe en ooreenstemmende wyne. Die waterstreseffek op meeste van die fenoliese parameters gemeet in druiwe en wyn was nie so prominent soos dié van die lowermanipulasie behandeling nie. Hierdie studie het ons begrip verbeter van hoe ‘n verbetering van die lower mikroklimaat van Shiraz gereflekteer kan word op die fenoliese samestelling van die wyn, saam met ‘n potensiële belangrike effek van oesdatum. Op grond van hierdie basis is dit dus moontlik om ‘n spesifieke wynstyl te verkry. Oes by ‘n ryp stadium, byvoorbeeld, kan die produksie van wyn met ‘n hoër kleurdigtheid en frankheid tot gevolg hê, terwyl onryp druiwe wyne met hoër vlakke van waarneembare vars bessiekenmerke tot gevolg kan hê. Verlaging van lowerdigtheid kan veral die frankheid en mondgevoel van wyne gemaak van druiwe blootgestel aan waterstres, verbeter.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Chenin blanc wine is of economic importance to South Africa and a range of diverse dry and semi-dry wines are locally produced in this genre. Currently, the use of three distinctly different style names, each aimed at providing consumers with information about the flavour of the wines, is encouraged by the South African (SA) wine industry. The styles are fresh and fruity (FF), rich and ripe unwooded (RRUW) and rich and ripe wooded (RRW). Feedback from retail sectors over the past few years, however, repeatedly suggested that the style names are perceived as confusing by SA consumers. This master study was undertaken to re-evaluate the FF, RRUW and RRW style classification, based on both the volatile fermentation-derived aroma composition and the sensory attributes of a set of wines containing all the styles under investigation. For the purposes of chemical profiling, a set of 105 commercial Chenin blanc wines, selected to be representative of these three styles and originating from the major SA wine producing areas, were analysed by Gas Chromatography (GC) to quantify fermentation-derived volatile aroma compounds in the wines. ANOVA performed on the chemical data showed that 29 compounds represent significant differences between at least two of the 3 styles (FF, RRUW and RRW). Principal component analysis (PCA) of the volatile compounds showed a large degree differentiation between FF and RRW wine styles, however, RRUW wine styles overlapped with the other two styles. Considering vintage effects, ANOVA indicated no significant differences within FF (vintages 2009 and 2010) and RRW (vintages 2008 and 2009) styles, whereas only 2 esters and 4 terpenes showed significant differences between the three wine producing regions investigated for this purpose, Paarl/Wellington, Breede River and Stellenbosch. Volatile aroma compounds generated for Chenin blanc were included in the Winetech database consisting of the most important cultivars of South Africa. Combining the data for the volatiles for Chardonnay and Sauvignon blanc from this database and the data for Chenin blanc obtained in this study, a PCA indicated a clear separation between Chenin blanc and the other two white cultivars. Sensory evaluation of the style classification was done by two separate sensory tests. Firstly, a sorting task was performed by wine industry experts to categorise 21 Chenin blanc wines (FF, RRUW and RRW) based on their similarity. The results showed a differentiation between FF and RRW styles, however, RRUW was mostly classified together with FF wines. This indicated a possible continuum between the three styles, as opposed to three distinct different categories, currently suggested by the style names. The second sensory analysis test, Descriptive Sensory Analysis (DSA), was performed by a trained panel to generate sensory profiles for 42 wines. ANOVA of the flavour attribute intensities between different styles once again showed significant differences between FF and RRW, with RRUW wines forming a continuum between the FF and RRW styles. These results provide valuable information that could be used by the wine industry for labelling purposes. / AFRIKAANSE OPSOMMING: Chenin blanc is van ekonomiese belang vir Suid Afrika en ‘n wye reeks droë en semi-droë wyne word plaaslik geproduseer in hierdie kategorie. Tans word die gebruik van drie duidelike verskillende stylbenamings, elkeen daarop gemik om aan die verbruiker inligting te verskaf oor die geur van die wyn, deur die Suid Afrikaanse (SA) wynindustrie aangemoedig. Die style is vars en vrugtig, ryk en ryp ongehout en ryk en ryp gehout. Terugvoer van die handelssektor oor die afgelope aantal jare, het daarop gedui dat die stylbenamings tot verwarring onder SA verbruikers lei. Hierdie meestersstudie is onderneem om die stylklassifikasie, vars en vrugtig, ryk en ryp ongehout en ryk en ryp gehout, te her-evalueer op grond van die vlugtige aroma komponente wat tydens die fermentasie proses gevorm word, asook die sensoriese eienskappe van ‘n verteenwoordigende stel wyne van elk van die style wat ondersoek is. Vir die doel van die chemiese profilering, is ‘n stel van 105 kommersiële wyne, wat geselekteer is om verteenwoordigend te wees van die drie style ondersoek en ook afkomstig is van die vernaamste SA wynproduserende streke, gebruik. Die wyne is met behulp van gas chromatografie ontleed om die vlugtige komponente wat van die fermentasie proses afkomstig is, te kwantifiseer. Die analise van variansie, het getoon dat 29 komponente statisties beduidend verskil het tussen die drie style. Hoofkomponent analise van die vlugtige komponente, het getoon dat die vars en vrugtige wyne en ryk en ryp gehoute wyne, duidelik onderskeibaar was van mekaar op grond van die vlugtige data, maar die ryk en ryp ongehoute wyne het met die ander twee style oorvleuel. In terme van oesjaar effekte, was daar geen beduidende verskille in die aroma profiele van die vars en vrugtige styl (oesjare 2009 en 2010) en ryk en ryp ongehoute styl (oesjare 2008 en 2009) nie, terwyl die konsentrasie van slegs twee esters en 4 terpene statisties beduidend verskil het tussen die wynproduserende streke Paarl/Wellington, Breederivier en Stellenbosch. Resultate van die gekwantifiseerde vlugtige komponente is in die databasis van Winetech gevoeg, waar die konsentrasies van soortgelyke komponente van die vernaamste SA wynkultivars reeds vervat is. Hoofkomponent analises van die gekombineerde resultate vir Chenin blanc, Chardonnay en Sauvignon blanc wyne, het getoon dat daar ‘n duidelike verkil tussen Chenin blanc en die ander twee wit wynkultivars was. Die sensoriese evaluerings is uitgevoer deur van twee verskillende metodes gebruik te maak. Eerstens is 21 wyne (met al drie style verteenwoordig) deur wynindustrie eksperts gesorteer op grond van die waargenome eendersheid van die onderskeie wyne en die resultate is grafies geprojekteer. Die resultate het getoon dat daar ‘n duidelike verskil waargeneem is deur die assessors tussen die vars en vrugtige styl en ryk en ryp gehoute styl. Die ryk en ryp ongehoute wyne het in die analises meer met die vars en vrugtige style geassosieer, as die ryk en ryp gehoute wyne. Die tweede sensoriese metode is uitgevoer deur sensoriese paneel wat vir die doel van hierdie studie opgelei is om die geur eienskappe van 42 wyne (al drie style verteenwoordig) te profileer. Analise van statistiese beduidende verskille tussen die voorkoms van die geurkomponente en hul intensiteite vir elke styl, het weereens aangedui dat daar ‘n kontinuum bestaan tussen die style. Hierdie resultate kan van waarde vir die wynindustrie wees in besluite rakende etikettering.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Wine fermentation is a continuously changing biological process whereby the raw product, grape juice is transformed into a high value product wine. In an ideal situation the fermentation kinetics of batch fermentations should follow the same trend over time. This however is not the case in industrial wine fermentations where significant batch-to-batch variation is present. The time trajectories of fermentation processes are therefore often unpredictable in absolute terms. The monitoring of substrate (sugar) and product concentrations (ethanol) as well as other quality parameters during a wine fermentation, is therefore of extreme importance to ensure effective control and management of wine fermentation processes. Conventional methods for fermentation monitoring are however costly, time consuming and often unreliable. For these reasons the modern wine industry requires rapid, reliable, non-destructive monitoring techniques which would meet the criteria of providing critical real-time process information that is displayed in easily interpretable graphical format, in order to ensure the highest quality and continuous consistency throughout all the stages of a process. This research study in particular, addressed the current need for alternative fermentation monitoring strategies that meet these criteria, by evaluating the potential use of spectroscopy as an analytical technique for fermentation monitoring. The overall objective of this study was to use chemometric modelling of information obtained by Fourier transform mid-infrared (FT-MIR) and near-infrared (FT-NIR) spectroscopy, to quantitatively and qualitatively monitor both alcoholic (AF) and malolactic fermentation (MLF) processes. Towards this objective 11 batch fermentations elaborated with Oenococcus oeni and Lactobacillus plantarum strains in respectively a co-inoculation and sequential inoculation scenario, were sampled and analysed at regular time intervals with FT-MIR and FT-NIR spectroscopy and enzymatic reference methods during 2011. Samples were also analysed by gas chromatography flame ionisation detection (GC-FID) and mass spectrometry (GC-MS) at two critical stages during fermentation, namely 50% completion of MLF and 100% completion of MLF, in order to obtain a profile of the evolution of the aroma compounds associated with each inoculation scenario. Three clearly defined research objectives were set for this project. The first objective involved the expansion of the existing quantitative platform for fermentation monitoring. Towards the outcomes of this objective, partial least squares (PLS) calibration models for prediction of malic acid and lactic acid in fermenting must and wines elaborated in our study, were established, based on the MIR and NIR spectra. The models showed excellent predictive abilities in independent test set validation. This outcome made a significant contribution to our existing PLS calibration capacity, particularly towards monitoring of MLF. Quantitative data obtained with the PLS models were also used to graphically project the rate of AF in the different batches, by non-linear fitted regression plots that easily visualised the overall patterns of sugar and ethanol metabolism in the different fermentations. The second research objective involved the qualitative monitoring of fermentations. This approach used FT-MIR and FT-NIR spectra together with chemometrics to identify trends between the different fermentation treatments. Principal component analysis (PCA) clearly projected the time trend from the onset of fermentation, through AF and MLF. No unique bacterial trend was however observed with spectroscopy. These results illustrate the potential of these techniques to be used for modelling of fermentations in industrial situations, through providing critical information about the evolution of the process. Furthermore, these techniques provide tools for identifying problematic and deviating fermentations. A spectral conformity test based on simple calculations of the standard deviation between the absorbance at each recorded wavenumber in the spectra, further confirmed identification of the critical fermentation stages. This technique by-passes the need for spectral interpretation and is a very useful addition, particularly from the industry perspective, to the portfolio of methods established in this study. The third research objective adressed the need to evaluate the possibility to discriminate between the different process stages and LAB treatments using univariate (ANOVA) and multivariate chemometric techniques such as PCA, PLS discriminant analysis (PLS-DA) and Soft Independent Modelling of Class Analogy (SIMCA) for possible future interpretative and classification purposes. The exploratory tool of PCA was used to investigate the similarities and differences between the chemical footprints of the different treatments. PCA showed clear differentiation between the two process stages using chemical quantified data. Differentiation between the LAB treatments was visible with PCA, showing a more prominent separation at 50% completion of MLF. Furthermore the ability of spectroscopy for potential classification was shown using PLS-DA and SIMCA. This profiling study can be seen as a preliminary study setting the ground work for further in-depth research into the profiling of different LAB treatments and inoculation strategies. / AFRIKAANSE OPSOMMING: Wyngisting is ‘n voortdurend veranderende biologiese proses waarvolgens die rou produk, druiwesap, in ‘n produk van hoë waarde, naamlik wyn, verander word. Onder ideale omstandighede sou die gistingskinetika van lotgistings dieselfde tendens oor tyd volg. Dit is egter nie die geval in industriële wyngistings nie, waar noemenswaardige wisseling van lot tot lot teenwoordig is. Die tydsbaan van gistingsprosesse is dus in baie gevalle in absolute terme onvoorspelbaar. Die monitering van substraat- (suiker) en produkkonsentrasies (etanol), sowel as ander kwaliteitsparameters tydens ‘n wyngisting, is van die uiterste belang om doeltreffende beheer en bestuur van wyngistingsprosesse te verseker. Konvensionele metodes vir die monitering van gisting is egter duur, tydrowend en ook soms onbetroubaar. Om hierdie redes vereis die moderne wynbedryf vinnige, betroubare en nie-destruktiewe moniteringstegnieke wat aan die kriteria sou voldoen vir die verskaffing van kritiese, intydse prosesinligting wat in ‘n maklik interpreteerbare grafiese formaat vertoon word om die hoogste kwaliteit en voortdurende konsekwentheid tydens al die stadia van ‘n proses te verseker. Hierdie navorsingstudie het in besonder die huidige behoefte aan alternatiewe gistingsmoniteringstrategieë wat aan hierdie kriteria voldoen, aangespreek deur die potensiële gebruik van spektroskopie as ‘n analitiese tegniek vir die monitering van gisting te evalueer. Die oorhoofse doelwit van hierdie studie was die chemometriese modellering van inligting wat met behulp van Fourier transform middel-infrarooi (FT-MIR) en naby-infrarooi (FT-NIR) spektroskopie verkry is om die prosesse van alkoholiese en appelmelksuurgisting (AMG) kwantitatief en kwalitatief te monitor. Ten einde hierdie doelwit te bereik, is 11 lotgistings met Oenococcus oeni en Lactobacillus plantarum rasse uitgevoer in scenario’s van ‘n gesamentlike inokulasie en opeenvolgende inokulasie onderskeidelik. Monsters van hierdie gistings is met gereelde tydintervalle in 2011 geneem en analises is met FT-MIR en FT-NIR spektroskopie en ensiematiese verwysingsmetodes gedoen. Monsters is ook met gaschromatografiese vlam ionisasie-opsporing (GC-FID) en massaspektrometrie (GC-MS) op twee kritiese stadia tydens gisting geanaliseer, naamlik toe AMG 50% en 100% voltooid was, om ‘n profiel te verkry van die evolusie van die aromaverbindings wat met elke inokulasie-scenario verband hou. Drie duidelik gedefinieerde navorsingsdoelwitte is vir hierdie studie bepaal. Die eerste doelwit het die uitbreiding van die bestaande kwantitatiewe platform vir gistingsmonitering behels. Hiervoor is gedeeltelike kleinstekwadraat [partial least squares (PLS)] kalibrasiemodelle vir die voorspelling van appelsuur en melksuur in die gistende mos en wyne in ons studie op die basis van MIR- en NIR-spektra bepaal. Die modelle het in onafhanklike geldigheidsbepaling van die toetsstel uitstekende voorspellingsvermoëns getoon. Hierdie uitkoms het ‘n noemenswaardige bydrae gemaak tot ons bestaande PLS kalibrasiekapasiteit, veral om AMG te monitor. Die kwantitatiewe data wat met die PLS-modelle verkry is, is ook gebruik om die tempo van alkoholiese gisting in die verskillende lotte grafies uit te beeld deur middel van kromlynige passing van regressiepersele, waarmee dit maklik was om die algehele patrone van suiker- en etanolmetabolisme in die verskillende gistings te visualiseer. Die tweede navorsingsdoelwit het die kwalitatiewe monitering van gistings behels. Hierdie benadering het FT-MIR en FT-NIR spektra tesame met chemometrie gebruik om tendense tussen die verskillende gistingsbehandelings te identifiseer. Hoofkomponentanalise [principal component analysis (PCA)] het duidelik die tydtendens vanaf die aanvang van gisting, deur alkoholiese gisting en AMG, geprojekteer. Geen unieke bakteriese tendens is egter met spektroskopie waargeneem nie. Hierdie uitslae illustreer die potensiaal van hierdie tegnieke om vir die modellering van gistings in industriële situasies gebruik te word deur kritiese inligting oor die evolusie van die proses te verskaf. Verder verskaf hierdie tegnieke gereedskap vir die identifikasie van problematiese en afwykende gistings. ‘n Spektrale gelykvormigheidstoets gebaseer op eenvoudige berekeninge van die standaardafwyking tussen die absorbansie by elke aangetekende golfgetal in die spektra het ook die identifikasie van die kritiese gistingstadia bevestig. Hierdie tegniek omloop die noodsaak vir spektrale interpretasie en is ‘n baie nuttige byvoeging tot die portefeulje van metodes wat in hierdie studie bepaal is, veral vanuit ‘n bedryfsperspektief. Die derde navorsingsdoelwit het die behoefte aangespreek om tussen die verskillende prosesstadia en melksuurbakterie-behandelings te onderskei deur gebruik te maak van eenvariant- (ANOVA) en meervariant- chemometriese tegnieke soos PCA, PLSdiskriminantanalise en sagte onafhanklike modellering van klasanalogie [soft independent modelling of class analogy (SIMCA)] vir moontlike toekomstige verklarende en klassifikasiedoeleindes. Die ondersoekende gereedskap van PCA is gebruik om ooreenkomste en verskille tussen die chemiese voetspore van die verskillende behandelings te ondersoek. PCA het duidelike differensiasie tussen die twee prosesstadia getoon op grond van chemies gekwantifiseerde data. Differensiasie tussen die melksuurbakterie-behandelings was met PCA sigbaar, met ‘n meer prominente skeiding teen 50% voltooide AMG. Verder is die vermoë van spektroskopie vir potensiële klassifikasie met PLS-diskriminantanalise en SIMCA getoon. Die profielsamestelling in hierdie studie kan beskou word as ‘n voorlopige studie vir verdere diepgaande navorsing oor die profielsamestelling van verskillende melksuurbakteriebehandelings en inokulasiestrategieë. / The National Research Foundation and Winetech

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grape must is a complex medium, and during wine production numerous biochemical pathways and metabolic reactions are taking place simultaneously to produce a specific taste and aroma. Microorganisms, specifically yeast, play a key role in the formation of metabolites formed during alcoholic fermentation. Sauvignon blanc, a well studied grape cultivar, is known to have a versatile range of aroma profiles ranging from “green” to “tropical”. It has been broadly stated that a “green” Sauvignon blanc can be created in the vineyard and a “tropical” Sauvignon blanc can be created by selecting a specific yeast strain, and that the balance between “green” and “tropical” characters is essential for the final aroma profile. Except for grape-derived varietal aromatic compounds such as methoxypyrazines (green), volatile thiols (tropical) and monoterpenes (floral), yeast derived volatile compounds including esters, higher alcohols, fatty acids and carbonyl compounds will also contribute to the final wine aroma. The main aim of this study was to assess how viticultural treatment-derived differences in grape must, can impact on aroma production when this grape must is fermented with different commercial wine yeast strains. The viticulture treatment focused on light intensity modulated through canopy treatment. Volatile aroma differences were compared for canopy and yeast treatments, specifically focusing on the fermentation derived bouquet (esters, higher alcohols, volatile fatty acids, carbonyl compounds and monoterpenes). Results showed significant differences between initial must compositions, including titratable acidity, malic acid and yeast assimilable nitrogen. The volatile aroma compounds were also significantly impacted although no noticeable effect on the overall fermentation kinetics was observed. Depending on the yeast strain differences in volatile compounds varied. A clear vintage effect is noticeable between volatile compounds affected by the treatments. Data generated in 2012 shows clear differences between ethyl- and acetate esters and could clearly be grouped according to yeast strain through multivariate analysis. Sensory evaluation results could clearly be distinguished according to canopy treatment and to a lesser degree according to yeast strain used. This indicates that although yeast has a more prominent impact on the fermentative bouquet that develops during alcoholic fermentation the overriding aroma is primarily derived from grape-derived metabolites which can be manipulated by canopy treatments. None the less the difference in fermentation bouquet does add to the complexity of the wine especially in the case of fermentation derived “tropical” aromas including guava and passion fruit. In some cases where shaded grapes had higher ester concentrations, the resultant wine also had higher aroma quality. This study has contributed to a better understanding of the complex relationships between canopy manipulation and yeast selection on aroma formation. The analysis of volatile aroma alone however is not enough to understand the final perception of wine taste and further indepth studies of the viticultural and oenological factors is needed. In particular, this project has focused on a single vineyard over only two vintages. The general validity of the conclusions derived from this study therefore will require additional data sets. / AFRIKAANSE OPSOMMING: Druiwemos is ‘n komplekse medium en tydens wynbereiding is daar verskeie biochemiese weë en metaboliese reaksies wat gelyktydig plaasvind om ‘n spesifieke smaak en aroma te produseer. Mikro-organismes, veral gis, speel ‘n sleutelrol in die vorming van metaboliete tydens alkoholiese gisting. Sauvignon blanc, ‘n goed bestudeerde druifkultivar, besit ‘n veelsydige reeks aromaprofiele wat wissel van “groen” tot “tropies”. Oor die algemeen word dit voorgehou dat ‘n “groen” Sauvignon blanc in die wingerd geskep word, terwyl ‘n “tropiese” Sauvignon blanc geskep kan word deur ‘n spesifieke gisras te selekteer, en die balans tussen “groen” en “tropiese” karakters is noodsaaklik vir die finale aromaprofiel. Behalwe vir druifafgeleide kultivarafhanklike aromatiese verbindings soos metoksipirasiene (groen), vlugtige tiole (tropies) en monoterpene (blomagtig), sal gisafgeleide vlugtige komponente, waaronder esters, hoër alkohole, vetsure en karbonielverbindings, ook tot die finale wynaroma bydra. Die hoofdoelwit van hierdie studie was om te bepaal hoe verskille in druiwemos wat afkomstig is van wynkundige behandeling ‘n impak op aromaproduksie kan hê wanneer hierdie druiwemos met verskillende kommersiële wyngisrasse gegis word. Die wynkundige behandeling het gefokus op ligintensiteit wat deur lowerbehandeling gereguleer is. Vlugtige aromaverskille is op grond van lower- en gisbehandelings vergelyk, met ‘n spesifieke fokus op die gistingsafgeleide boeket (esters, hoër alkohole, vlugtige vetsure, karbonielverbindings en monoterpene). Die resultate het beduidende verskille getoon tussen aanvanklike mossamestellings, waaronder titreerbare suurheid, appelsuur en gis-assimileerbare stikstof. Daar was ook ‘n noemenswaardige impak op die vlugtige aromaverbindings, hoewel geen merkbare effek op die algehele gistingskinetika waargeneem kon word nie. Die verskille in vlugtige verbindings het gewissel op grond van die gisras. ‘n Duidelike oesjaareffek was merkbaar tussen vlugtige verbindings wat deur die behandelings geaffekteer is. Data wat in 2012 gegenereer is, toon duidelike verskille tussen etiel- en asetaatesters en kon duidelik m.b.v. meervariantanalise volgens gisras gegroepeer word. Die resultate van die sensoriese evaluering kon duidelik volgens lowerbehandeling onderskei word, en tot ‘n mindere mate volgens die gisras wat gebruik is. Dít dui daarop dat hoewel gis ‘n meer prominente impak het op die gistingsboeket wat tydens alkoholiese gisting ontwikkel, is die oorheersende aroma hoofsaaklik afgelei van druifafgeleide metaboliete wat deur lowerbehandelings gemanipuleer kan word. Nietemin dra die verskil in gistingsboeket by tot die kompleksiteit van die wyn, veral in die geval van gistingsafgeleide “tropiese” aromas, insluitend koejawel en grenadella. In sommige gevalle waar beskadude druiwe hoër esterkonsentrasies gehad het, het die gevolglike wyn ook ‘n hoër aromakwaliteit gehad. Hierdie studie dra by tot ‘n beter begrip van die effek van die komplekse verhoudings tussen lowermanipulasie en gisseleksie op aromavorming. ‘n Analise van vlugtige aroma alleen is egter nie voldoende om die finale persepsie van wynsmaak te begryp nie en bykomende diepgaande studies van die wingerdkundige en wynkundige faktore word benodig. Hierdie projek het in die besonder gefokus op ‘n enkele wingerd oor slegs twee oesjare. Die algemene geldigheid van die afleidings wat van hierdie studie gemaak word, sal dus bykomende datastelle vereis. / The National Research Foundation and Postgraduate Merit Bursary for financial support

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The quality of wine is largely dependent on environmental conditions and recent studies have therefore focused on agricultural practices in terms of water, soil and biodiversity conservation. The industry aims to create sustainable practice and to protect the natural resources available. Sources of potential grape contamination include: vehicle pollution, pesticides, bushfires and wood preservatives used for trellising systems. The latter have come to the attention of the South African wine industry (e.g. creosote and Copper Chromium Arsenate (CCA) products) as they may have consequences for the environment and wine quality. Creosote is a known pollutant of soil and ground water and the volatile fraction has been monitored in air. Plants may also accumulate polycyclic aromatic hydrocarbons (PAHs), which constitute up to 85% of the mass of creosote, and of which some have been proven carcinogenic. Because of the health risks associated with it, creosote has therefore been restricted for use in most applications in Europe, and also in the United States, Canada and Australia. This study focussed on the sensorial and chemical analyses of environmental and wine samples taken from the area around the creosote stockyard to determine accumulation of creosote-derived compounds. Environmental samples were collected and analysed at different distances from the affected area, over two vintages. Wines were made from grapes grown in vineyard blocks adjacent to the stockyard, to determine the effect of distance and skin contact during alcoholic fermentation treatments on wine taint. A sensory panel was trained for descriptive analysis to determine the intensity of the taint. Analytical methods were developed for the analysis of volatile organic compounds (VOCs) by gas chromatography mass spectrometry (GC-MS) and polycyclic aromatic hydrocarbons by high performance liquid chromatography with diode array detection (HPLC-DAD). The sensory results obtained showed white and rosé wines were perceived as clean, whilst the red wines were associated with burnt rubber/tar taint. The perceived taint decreased as distance from the stockyard increased. Wines made from the Cabernet Sauvignon blocks adjacent to the stockyard also showed an increase of taint with the increase of skin contact. Chemical data obtained showed that the taint consisted of a complex mixture of compounds, each with its own pattern of retention within the vineyard and wine. Only m-cresol was found above odour threshold, and only in red wines. The synergistic effect of some compounds may lead to an increase in the perceived taint. Berries and leaves had higher concentrations of volatile compounds than wines. Leaf contamination varied and followed the general trend in literature where the plants with high lipid content and exposed leaf area were the most contaminated. There may be other compounds present in creosote emissions, aside from those investigated here, with sensory attributes related to the taint found in wines. These compounds are styrene, indene, benzene, toluene, isoquinoline and quinoline and should be included in further investigations. From the PAH analysis of environmental samples and wines, it is evident that the samples closest to the stockyard were affected the most. The contamination varied with the depth of the soil: some of the heavier compounds were found in the samples taken from the deeper levels, whilst nearly all other target compounds were present in the top layer of soil. The concentrations found in the environmental samples were lower than reported in literature. Wines had few PAHs present, but at much higher concentrations than is allowed by EU legislation. From this study it is evident that the stockyard had negative effects on the surrounding environment in terms of sensory and chemical contamination. Recommendations include area rehabilitation by means of bioremediation to protect resources and ensure sustainable and safe production of crops. Industrial emissions should also be regulated and restricted in agricultural areas. Furthermore the use of creosote should be revised, and prohibited for agricultural use. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn is grootliks afhanklik van die omgewingstoestande. Daarom fokus verskeie onlangse studies op landbou aktiwiteite en die invloed daarvan op die omgewing in terme van water, grond en biodiversiteit bewaring. The wyn industrie se doelwitte sluit volhoubare praktyke in, wat die natuurlike hulpbronne bewaar en beskerm. Druiwe kan deur middel van die volgende bronne besoedel word: brandstof uitlaatgasse, insekdoders, veldbrande, gifstowwe wat gebruik word vir houtperservering wat dan verder aangewend word vir opleistelsels. Houtperservering (Koper chroom arsenaat (CCA) en kreosoot) het veral in die laaste paar jaar onder aandag gekom in die wynbedryf van Suid-Afrika as gevolg van die invloed daarvan op die omgewing en die uiteindelike wynkwaliteit. Kreosoot is ‘n gekende gifstof wat verantwoordelik is vir grond en grondwater besoedeling en wat gemonitor word in die atmosfeer. Plante akkummuleer ook poli-sikliese aromatiese hidro-koolstowwe (PAHs), wat to 85% uitmaak van die massa van kreosoot. PAHs is karsinogenies en dus is daar baie navorsing op die molekules reeds gedoen. Die gesondheidsrisikos gepaardgaande met kreosoot het gelei tot die streng geregulasies tans ingestel in Europa, die Verenigde State, Kanada en Australië. Hierdie studie het gefokus op die sensoriese en chemiese analises van omgewings- en wyn-monsters geneem van die omliggende area van die kreosoot palewerf om die akkumulasie van kreosoot-afgeleide-verbindings. Omgewingsmonsters was versamel en geanaliseer om verskillende afstande vanaf die bron van besoedeling (palewerf) te ondersoek oor ‘n twee jaar periode. Wyne is gemaak van die druiwe wat afkomstig is van die blok aangeplant langs die palewerf. Die wyne is ondersoek in terme van afstand vanaf die kreosoot bron asook oenologiese invloede, dopkontak gedurende alkoholiese fermentasie, op die kontaminasie beskryf in wyn. Die wyne is ook oor ‘n twee jaar periode voorberei en sluit die 2011 en 2012 seisoen in. ‘n Sensoriese paneel is opgelei om die beskrywende analises op die wyn uit te voer met die doel om die intensiteit van kontaminasie te identifiseer. Analitiese metodes is ook ontwikkel vir die analise van vlugtige organiese verbindings(VOCs) met gas chromatografie-massa spektrometrie (GCMS) en poli-sikliese aromaties hidro-koolstowwe (PAHs) met hoë druk vloeistof chromatografie. Die sensoriese resultate bekom het wit en rosé wyne as skoon laat blyk, terwyl rooi wyne meer geassosieer was met die gebrande rubber/ teer afgeur beskrywing. Die waargeneemde afgeur het afgeneem soos wat die afstand vanaf die palewerf toegeneem het. Wyne gemaak van die Cabernet Sauvignon blokke langsaan die palewerf het ook ‘n toename in die afgeur gehad met ‘n toename in dopkontak. Chemiese data bekom beeld uit dat die afgeur uit ‘n komplekse mengsel van verbindings bestaan, elk met sy eie patroon van verspreiding en verbinding in die wingerd as ook in die wyn. Net m-kresol was gevind bo die reuk drumpel, dit het ook net in rooi wyne voorgekom. Die sinergistiese effek van die verbindings mag egter bydra tot die waargeneemde afgeur. Druiwekorrels en blare het hoër konsentrasies van die vlugtige verbindings gehad as wat gemeet is in die wyne. Blaar kontaminasie het ook baie gewissel en het ooreengestem met die algemene tendens wat in literatuur beskryf is, naamlik dat plante met ‘n hoër lipid inhoud en grootter blaar oppervlak die meeste gekontamineer word. Daar mag egter nog baie ander verbindings bydra tot die waargeneemde afgeur gevind in die wyn. Spesifieke verbindings wat wel ‘n rol kan speel in kontaminasie en wat voorkom in die vlugtige gedeelte van kreosoot is styreen, indeen, benzeen, tolueen, isoquinoleen die vlugtige verbindings van kreosoot. Die verbindings moet ingesluit word vir verdere studies wat gedoen word op die kreosoot geassosieerde afgeur. Die PAHs analise op die omgewingsmonsters en wyne het gelei tot die bevestiging dat die naasliggende omgewing die meeste geaffekteer is. Die kontaminasie wissel in terme van die diepte in die grond wat die gifstowwe voorkom: die swaarder molekulêre verbindings is tot in die dieper vlakke waargeneem terwyl al die gemete verbindings in die boonste lae teenwoordig was. Die vlakke wat waargeneem is in dié studie is egter laer as wat voorheen in literatuur gevind is in ‘n kreosoot geaffekteerde omgewing. Wyn het PAHs teenwoordig gehad, alhoewel slegs twee verbindings gemeet is, het dit in hoër vlakke voorgekom as wat sekere Europese regulasies as toelaatbaar spesifiseer. Vanaf die studie resultate blyk dit, dat die palewerf se negatiewe invloed op die omliggende omgewing beide meetbaar was in sensories en chemiese kontaminasie. Voorstelle sluit onder andere die rehabilitasie van die omliggende omgewing deur middel van bioremediasie in. Om sodoende die natuurlike hulpbronne in die area te bewaar asook om volhoubare en veilige verbouing van gewasse te verseker. Industriële besoedeling en afval moet ook gereguleer word en beperk word in landbou areas. Verder moet die gebruik van kreosoot heroorweeg word en strenger regulasies moet in plek gestel word om aan internastionale standaarde te voldoen. / Financial support from Winetech, Trip and UFS

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, 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

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as antifungal agents in agricultural biotechnology. These peptides have a broad spectrum antifungal activity and play a vital role in the innate immune system of plants. Plant diseases caused by fungi are a major contributor to the decrease in the quality and safety of agricultural products. Due to the dangerous effects and negative environmental impact of pesticides, an effective, safe, natural and durable method to control crop pathogens has therefore become one of the major concerns in modern agriculture. Although these peptides are promising and attractive candidates, their precise mechanism of action is to date still unknown. Several common observations have been made. These include the antagonistic effect of cations on the activity of plant defensins. It is of vital importance to understand the underlying mechanism of the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate the possible contribution to defence reactions against microorganisms in planta. To this end we set out to characterize the effect of cations in the form of biological salts, NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition, morphological effects and permeabilization. In order to perform these characterization experiments, a production method resulting in a greater yield and involving simple and rapid purification methods was required. Heliophila coronopifolia peptides have previously been produced in a bacterial system, however the purification methods were tedious resulting in poor yields. Pichia pastoris was selected as production system as several other plant defensins have been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc- AFP4 was, however, not produced correctly, due to a post-translational modification event leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be produced by replacing the production buffer with a reduced ionic buffer. The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was evaluated by circular dichroism spectroscopy. These cations induced a conformational change in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea, however, characteristic hyperbranching and other morphogentic effects were still visible. Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not influenced by the addition of cations, however it was in fact increased to up to 10-fold. However, since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological salts indicates that permeabilization is not the sole activity responsible for growth inhibition caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex MOA. This is the first known report of the investigation of the influence of cations on the structure of plant defensin peptides. It is clear that cations induce a secondary structural conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this peptide. This study provides significant progress towards the structure-function analysis of plant defensins. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te evalueer. Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig word deur die produksiebuffer te vervang met ‘n lae-ionise buffer. Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het ‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT), THRIP and Winetech for financial assistance.