Investigating polyol and acetic acid metabolism in wine related non-Saccharomyces yeasts

Thesis (MScAgric)--Stellenbosch University, 2017.ENGLISH SUMMARY: Glycerol is the main polyol produced in Saccharomyces cerevisiae not only to counterbalance osmotic pressure but also to adjust redox balance. Incidentally, it may also contribute to the smooth mouthfeel of wine. Whereas glycerol is closely linked to acetic acid production in S. cerevisiae, this correlation is not as clear in non-Saccharomyces yeasts (particularly Torulaspora delbrueckii). Additional polyols - which function as stress protectants and could potentially influence wine mouthfeel - have been reported in wine but the producing yeasts were never isolated. Lachancea thermotolerans, Starmerella bacillaris and T. debrueckii have been recently described as producing other polyols in addition to glycerol with the latter producing the highest amounts. However, the enzyme assays used were limited to polyol detection in combination. Thus, the aim of this study was to optimize chromatography-based methods for the separation of polyols and to investigate the production of these compounds in non-Saccharomyces yeasts under a variety of environmental conditions. Gas Chromatography-Mass Spectrophotometry was successful for the separation of polyols but only in fermentation samples with no residual sugars. Since non-Saccharomyces yeasts do not ferment to completion, other methods are required for the individual detection of polyols in order to follow production throughout fermentation. Our data show that in addition to glycerol, three T. delbrueckii strains increasingly produced similar amounts of D-sorbitol, D-arabitol and D-mannitol throughout fermentation. Furthermore with the exception of glycerol, T. delbrueckii produced higher amounts of polyols in grape must when compared to synthetic must. Whereas glycerol is limited to NADH recycling, these additional polyols may increase the co-factor recycling pool in T. delbrueckii. Our data also show that D-sorbitol, D-mannitol and D-arabitol production was influenced by initial sugar concentration with the highest amounts detected for D-arabitol in T. delbrueckii. In contrast to D-arabitol which was produced at the highest amounts, D-mannitol and D-sorbitol were not induced by NaCl. It is possible that these compounds may have accumulated within the cell as a consequence of the osmotic gradients or mechanisms related to the prevention of ion toxicity as observed in literature. Polyol production was repressed in acetic acid media in this study and induced in ethanol supplemented media. The intake of acetic acid could have resulted in a change in redox balance and a reduced need for polyols as reported in literature. The presence of ethanol could have resulted in readjustment of polyol retention within the cell and release of polyols. Overall this study shows that non-Saccharomyces yeasts (particularly T. delbrueckii) are capable of polyol production. The amounts of polyols produced in some non-Saccharomyces yeasts may have a direct impact on wine but further investigations are required on this.AFRIKAANS OPSOMMING: Gliserol is die hoof poli-ol wat deur Saccharomyces cerevisiae geproduseer word, nie net om osmotiese druk teen te werk nie, maar ook om die redoksbalans aan te pas. Dit mag ook bydrae tot die gladde mondgevoel van wyn. Waar gliserol baie nou geskakel is met die asynsuur produksie in S. cerevisiae, is hierdie korrelasie nie so duidelik in nie- Saccharomyces giste (veral Torulaspora delbrueckii) nie. Ander poli-ole – wat optree as spanningsbeskermers en moontlik wyn mondgevoel kan verander- is voorheen geraporteer in wyn, maar die produserende giste is nooit ge-isoleer nie. Lachancea thermotolerans, Starmerella bacillaris en T.delbrueckii is onlangs beskryf as produsente van poli-ole anders as gliserol, met die laasgenoemde wat die hoogste aantal produseer. Alhoewel die ensiem toets wat gebruik is slegs poli-ole in kombinasie kon optel. Die doel van hierdie studie was om die chromatograaf-gebaseerde metode te optimiseer vir die skeiding van poli-ole en om die produksie van hierdie verbindings in nie-Saccharomyces giste onder ‘n variasie van omgewingstoestande te toets. Gas chromotograaf-massa spektrofotometrie was suksesvol vir die skeiding van poli-ole, maar slegs in monsters van fermentasies wat geen residuele suiker bevat nie. Aangesien nie-Saccharomyces giste nie tot droogheid fermenteer nie, word ander metodes benodig vir die individuele deteksie van poli-ole om die produksie gedurende fermentasie te volg. Ons data toon dat addisioneel tot gliserol, drie T.delbrueckii rasse toenemend soortgelyke konsentrasies van D-sorbitol, D-arabitol, en D-mannitol geproduseer het gedurende fermentasie. Met die uitsondering van gliserol, produseer T.delbrueckii ‘n hoër aantal van poli-ole in druiwe sap as in sintetiese mos. Waar gliserol beperk is tot NADH herwinning, mag hierdie ander poli-ole die ko-faktor herwinnings poel in T.delbrueckii verhoog. Die data wys ook dat D-sorbitol, D-mannitol en D-arabitol produksie beïnvloed word deur die oorspronklike suikerkonsentrasie, met die hoogste konsentrasie gevind vir D-arabitol in T. delbrueckii fermentasies. In kontras met D-arabitol wat in die hoogtse konsentrasies geproduseer word, is D-mannitol en D-sorbitol produksie nie deur NaCl ge-induseer nie. Dit is moontlik dat hierdie verbindings in die sel geakkumuleer het as ‘n nagevolg van die osmotiese gradient of meganismes verwand aan die voorkoming van ion vergiftiging soos in die literatuur bespreek. Poli-ool produksie was onderdruk in asynsuur media in hierdie studie en aangewakker in etanol aangevulde media. Die inname van asynsuur kon ‘n verandering in die redoksbalans tot gevolg gehad het en die en ‘n verlaging in die behoefte vir poli-ole soos in die literatuur bespreek. Die teenwoordigheid van etanol was moontlik verantwoordelik vir die aanpassing in die poli-ool retensie binne-in die sel en die vrystelling van poli-ool. Hierdie studie wys dat nie-Saccharomyces giste (veral T.delbrueckii) in staat is tot poli-ool produksie. Die aantal poli-ole wat deur sommige nie-Saccharomyces giste geproduseer word het moontlik ‘n direkte impak op wyn, maar verdere ondersoeke word benodig