Geographical distribution, characterisation and evaluation of Saccharomyces cerevisiae strains isolated from South African vineyards in the warmer, inland regions of the Western Cape

[摘要] ENGLISH SUMMARY: The natural or spontaneous fermentation of grape must sugars, by yeast that originate fromthe grapes and winery equipment, to ethanol, carbon dioxide and other minor, but importantmetabolites, produces wine. The early stages of fermentation are usually carried out by theyeast genera with low fermentative activity, such as Hanseniaspora, Kloeckera, Candida andPichia. An increase in the alcohol content of the wine causes the suppression of thesegenera, leaving Saccharomyces cerevisiae, which plays a significant and dominating role, tocomplete the fermentation process. For this reason S. cerevisiae is then generally acceptedand known as the wine yeast in the wine industry.Wines made in different areas from the same grape variety with apparently similar fruitcomposition and processing, often taste noticeably different. It may be that differences in themicroflora, naturally present in the two areas, play an important role in producing flavourdifferences in the wines. The genetic diversity of the yeast flora in nature was proven, by anumber of microbiological and oenological studies, to be greatly influenced by a series ofparameters, which among others includes the age of the vineyard, grape variety, viticulturaland oenological practices, geographic location and climatic conditions.This project, as part of a larger research programme described by Pretorius, Van derWesthuizen & Augustyn (1999), was directly aimed at isolating and characterising indigenousS. cerevisiae yeast strains from vineyards based in the warmer, inland wine regions of theWestern Cape in South Africa. Research by Van der Westhuizen, Augustyn & Pretorius(1999a) and Van der Westhuizen et al. (1999b) on the wine yeast strains present on grapesin the cooler, coastal areas of the same province enabled us to compare these strainsisolated from these two contrasting climatic zones on a molecular, physiological andbiochemical level.The ideal or ultimate aim of any such study would be to determine the entire sequence of thebases of the yeast nucleic acid, for each of these isolates. Molecular biological, physiologicaland biochemical techniques can, however, be used as indicators of the homology of thechromosomes of strains or isolates obtained. The specific aims and approaches to this studywere as follows: Firstly approximately 1 kg of grapes were sampled from each of the 19 sitesin the warmer, inland wine regions. Areas sampled included: Villiersdorp, Robertson (2 sites),Nuy, Du Toits Kloof, Bonnievale, Ashton, Montagu (2 sites), Rawsonville, Riebeeck-Kasteel(2 sites), Porterville (2 sites), Wolseley, Malmesbury (2 sites), Sianghoek and Tulbagh.These grapes were aseptically crushed and the must allowed to spontaneously ferment at15°C. Thirty single colonies per fermentation were isolated when the residual sugar was lessthan 4 g/l. Characterisation of these S. cerevisiae isolates was done using molecularbiological fingerprinting techniques such as electrophoretic karyotyping by pulsed field gelelectrophoresis [Contour-Clamped Homogenous Electric Field, (CHEF)] and RandomlyAmplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) using five specificOperon Kit C (OPC) primers. Comparison of chromosomal banding patterns per samplingsite revealed the presence of 30 S. cerevisiae strains hereafter designated W1-W30. Initiallyit was thought a total of 21 yeast strains (based on CHEF banding pattern results only) were isolated from the warmer, inland regions. However, by comparing the CHEF-DNA analysisand RAOP-PCR data it was possible to distinguish between all of the strains isolated, exceptW1 and W5. Comparing these chromosomal banding patterns to the banding patternscollected in the Nietvoorbij database revealed that no single strain recovered was indigenousto both the warmer areas of this study and the cooler areas sampled by Van der Westhuizenat al. (1999a, 1999b). Further comparison and characterisation of these strains by means ofvarious physiological and biochemical techniques such as the ability to flocculate; fermentvarious carbon sources; survive stress resistance at 55°C and -20°C by monitoring cellgrowth after 24 and 48 hours, respectively; and produce extracellular enzymes, wasdetermined. Even though no distinct differences could be observed in the physiological andbiochemical behaviour of strains isolated from the warmer, inland and cooler, coastal wineregions, it was shown that strains W1 and W5, which had the same DNA banding patterns,were in fact different as W1 was galactose negative and W5 was galactose positive. StrainW1 could also grow invasively into the agar medium whereas W5 could not. Thiscomparison revealed that 30 unique strains were in fact isolated from the warmer, inlandwine regions of the Western Cape. Investigations into possible survival mechanisms ofS. cerevisiae was done by evaluating their ability to form pseudohyphae/grow invasively.Results indicated that these factors could contribute to the organisms over-wintering ability,but clearly other mechanisms must also be involved. Molecular biological, physiological andbiochemical characterisation techniques should then be used in collaboration with each otherto obtain an overall, positive and extensive profile of any yeast species or strain. Theseresults also then prove invaluable in not only providing information, but also elucidating anypossible differences between strains isolated from different climatic zones.The fermentation potential of these 30 unique strains was evaluated and of these strainsthree are included in the breeding programme at the ARC-Institute for Fruit, Vine and Wine,Nietvoorbij, aimed at improving wine yeast.

[发布机构] Stellenbosch University