Characterisation of Wickerhamomyces anomalus and Kazachstania aerobia : investigating fermentation kinetics and aroma production

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Non-Saccharomyces yeasts have been studied extensively in the past two decades to use as catalysts for adjusting the aroma and chemical properties of wine. Many non-Saccharomyces yeasts dominate in grape must, but Wickerhamomyces anomalus and Kazachstania aerobia have recently been found to be more dominant in several musts in South Africa than what has been reported from other wine growing areas. It has been hypothesised that regional microflora can lead to a terroir specific wine. To further establish these claims, the impact of these non-Saccharomyces yeasts on the chemical profile and sensory perception of wine, in particular when present in high numbers, has yet to be fully elucidated. This study was designed to better characterise isolated strains of non-Saccharomyces species, determining its phenotypic space, as well as to assess their fermentation potential and volatile aroma compound production in synthetic and real grape must. Eight K. aerobia and thirteen W. anomalus isolates were used for characterisation. DNA based taxonomic differences between isolates were investigated using the Random Amplification of Polymorphic DNA (RAPD) method and phenotypic heterogeneity was established using stress assays to determine heat, saline, osmotic and oxidative stress tolerance. Phenotypically diverse K. aerobia and W. anomalus strains were then selected for co- and sequential fermentations with two S. cerevisiae strains, VIN13 and EC1118, in synthetic grape must. In addition, sequential culture fermentations were conducted in Sauvignon blanc grape must by individually pairing two strains of K. aerobia and two strains of W. anomalus with S. cerevisiae EC1118. Wine aroma compounds were quantified using GC-FID. RAPD analysis classified W. anomalus isolates into five distinct groups according to place of origin. Phenotypic variations were evident within and between the proposed strains as was exhibited by heterogeneous resistance to oxidative, saline and osmotic stresses compared to S. cerevisiae, VIN13. The K. aerobia isolates showed no marked genetic differences, although exhibiting slight variations in stress responses. During fermentation the non-Saccharomyces yeasts persisted for longer when S. cerevisiae was only inoculated after 48 hours, or at a lower density. The longer the non-Saccharomyces yeasts proliferated in the must the more pronounced was the effect on aroma production. Kazachstania aerobia yeasts did not achieve a high biomass compared to W. anomalus, but survived for longer in fermentation, especially in Sauvignon blanc grape must. Although W. anomalus displayed strong growth, it was inhibited by the growth of S. cerevisiae. Kazachstania aerobia and W. anomalus gave a unique aroma profile to the wines. The latter yeast produced high concentrations of ethyl acetate, while K. aerobia was characterised by increased acetic acid concentration. Most aroma compounds were increased in mixed culture fermentations, especially higher alcohols, with a significant increase in the esters 2-phenylethyl acetate by K. aerobia, and ethyl caproate and caprylate by W. anomalus. Although, as single cultures these yeasts did not ferment wines to dryness in synthetic grape must and only completed fermentation after 28 days in Sauvignon blanc grape must, they are capable of conferring favourable wine aroma when in association with S. cerevisiae strains with no risk of sluggish fermentation. This study provides a basis for future work on wine quality improvement through exploitation of non-Saccharomyces yeasts and gives insight to the possible impact of K. aerobia and W. anomalus present in grape must in a South African context.AFRIKAANS OPSOMMING: Nie-Saccharomyces giste is in die afgelope twee dekades omvattend bestudeer om gebruik te word as katalisators vir die aanpassing van aroma en chemiese eienskappe van wyn. Baie nie-Saccharomyces giste domineer in druiwemos, maar onlangs is gevind dat Wickerhamomyces anomalus en Kazachstania aerobia meer dominant in verskeie druiwemos in Suid-Afrika is teenoor wat in ander wynbougebiede aangemeld is. Dit is voorgestel dat plaaslike mikroflora kan lei tot 'n terroir spesifieke wyn. Om hierdie stellings te evalueer, moet die impak van hierdie nie-Saccharomyces giste, veral wanneer hul in groot hoeveelhede teenwoordig is, op die chemiese profiel en sensoriese persepsie van wyn bepaal word. Hierdie studie is ontwerp om geïsoleerde gisrasse van nie-Saccharomyces spesies beter te karakteriseer, die fenotipiese ruimte te bepaal asook hul fermentasie potensiaal en aroma produksie in sintetiese en regte druiwemos vas te stel. Vir karakterisering, is agt K. aerobia en dertien W. anomalus isolate gebruik. DNA-gebaseerde taksonomiese verskille is ondersoek met die gebruik van die “Random Amplified Polymorphic DNA” (RAPD) metode, waarna fenotipiese heterogeniteit bepaal is met behulp van stres toetse deur hitte, sout, osmotiese en oksidatiewe stres toleransie te bepaal. Fenotipies diverse K. aerobia en W. anomalus gisrasse is daarna gekies vir ko- en sekwensiële fermentasies met twee S. cerevisiae gisrasse, VIN13 en EC1118, in sintetiese druiwe mos. Daarna is sekwensiële fermentasies in Sauvignon blanc sap uitgevoer deur individuele paring van twee gisrasse van K. aerobia en twee gisrasse van W. anomalus met S. cerevisiae EC1118. Aroma komponente is gekwantifiseer met die gebruik van GC-FID. RAPD-analise het W. anomalus isolate geklassifiseer in vyf afsonderlike groepe volgens plek van oorsprong. Fenotipiese variasies was duidelik waargeneem binne en tussen die voorgestelde gisrasse, soos voorgestel deur die heterogene weerstand teen oksidatiewe, sout en osmotiese spanning in vergelyking met S. cerevisiae, VIN13. Die K. aerobia isolate het geen merkbare genetiese verskille getoon nie, alhoewel effense variasies in stresreaksie waargeneem was. Gedurende fermentasie het die nie-Saccharomyces giste langer oorleef wanneer S. cerevisiae eers na 48 uur geïnokuleer was, of teen 'n laer digtheid. Hoe langer die nie-Saccharomyces giste oorleef het, hoe groter was die impak op aroma produksie. Alhoewel K. aerobia nie so ‘n hoë biomassa soos W. anomalus bereik het nie, het dit vir langer in fermentasie oorleef, veral in die Sauvignon blanc druiwe mos. Verder, alhoewel W. anomalus sterk gegroei het, was dit deur S. cerevisiae geïnhibeer. Kazachstania aerobia en W. anomalus het 'n unieke aroma profiel aan die wyne verleen. Laasgenoemde gis het hoë konsentrasies etielasetaat vervaardig, terwyl K. aerobia gekenmerk was deur 'n toename in asynsuur produksie. Die meeste aroma komponente het in die gemengde fermentasies toegeneem, veral die produksie van hoër alkohole, met 'n beduidende toename in die esters 2-fenieletiel asetaat deur K. aerobia, en etielkaprylaat en etielkaproaat deur W. anomalus. Alhoewel die wyne nie droog gegis was deur die giste as enkel kulture in sintetiese druiwe mos nie en eers ná 28 dae in Sauvignon blanc druiwe mos fermentasie voltooi het, was dit in staat om gunstige aromas aan die wyn te verleen en hou dit geen risiko vir slepende fermentasies in kombinasie met S. cerevisiae in nie. Hierdie studie bied 'n basis vir toekomstige werk oor die verbetering van wyngehalte deur die gebruik van nie-Saccharomyces giste en gee insig oor die moontlike impak van K. aerobia en W. anomalus wanneer teenwoordig in druiwe mos in 'n Suid-Afrikaanse konteks