From the vine to the table: Shaping the phenotype of wine using molecular genetic technology

From the vine to the table: Shaping the phenotype of wine using molecular genetic technology

Wine production, while considered an art form, owes an increasing debt to the latest molecular genetic technologies. Not only do these facilitate batch-to-batch consistency, they also enable the generation of novel strains of grape microflora. While the choice of grapes is important, so too is the interplay between extrinsic and intrinsic factors, and the technological decisions made at every step in the process. Together, these determine whether the finished product will be classed as a “vintage wine”, a “quaffing wine”, or “vinegar” [1, 2].

Several Saccharomyces and non-Saccharomyces yeast species, and lactic acid bacteria, feature in grape cultivation, wine production and spoilage. Saccharomyces (S.) cerevisiae, considered to be the “natural companion” of freshly-crushed grape juice (or grape must), converts grape sugar to alcohol and CO2, generates secondary metabolites, and converts grape aroma precursors to varietal wine aromas [3]. Non-Saccharomyces strains, also present in the initial fermentation process, are present on grapes, cellar equipment, in the cellar environment, and are also air- and insect-borne. These fall into one of three broad categories: (i) Aerobic Yeasts (e.g. Pichia spp., Debaryomyces spp., Rhodotorula spp., Candida spp., and Cryptococcus albidus); (ii) Apiculate Yeasts with low fermentative activity (e.g. Hanseniaspora uvarum, Hanseniaspora guilliermondii and Hanseniaspora occidentalis); and (iii) Yeasts with fermentative metabolism (e.g.  Kluyveromyces marxianus, Torulaspora delbrueckii, Metschnikowia pulcherrima and Zygosaccharomyces bailii).

Traditionally, non-Saccharomyces yeasts were thought to participate in grape must fermentation in a sequential manner and then gradually disappear as vigorous fermentation proceeded. Their disappearance was attributed to their slow growth, inhibition by SO2, low pH, high ethanol concentration, O2 deficiency, and by being out-competed by Saccharomyces spp. for nutrients. Recently, however, it was reported that the disappearance of desirable, non-Saccharomyces strains from grape must fermentation, could be abrogated by improvements in cellar hygiene and reduced SO2 usage. Improvements in molecular technologies have also made their detection easier [3]. The value of certain non-Saccharomyces strains cannot be overstated, as many of them contribute aromatic compounds to the finished product [3]. Non-Saccharomyces strains that cause grape spoilage, (e.g. Brettanomyces bruxellensis), are the arch enemy of the wine producer, but these too, can be kept in check by similar, diligent management of cellar hygiene, pH, SO2, temperature and nutrients [1, 3].  

Lactic acid bacteria participate in malolactic fermentation, which occurs predominantly in red wine production [1]. Three genera of lactic acid bacteria have been identified in grape must, namely (i) Lactobacillus, (ii) Oenococcus and (iii) Pediococcus, which are all capable of introducing defects into the wine, (e.g. acidification, bitterness or oiliness), or of causing spoilage [4].

Molecular typing of genetic variants of S. cerevisiae
Subtle genetic differences have emerged within S. cerevisiae isolates, and are thought to be an adaptive response to region-specific climatic conditions, soil composition, altitude, and cultivation techniques [5]. These genetic variants can be identified using a range of molecular biological techniques, including (i) Comparative Genomic Hybridization (CGH) arrays, (ii) Genome Sequencing and Functional Annotation, (iii) Pulsed Field Gel Electrophoresis (PFGE), (iv) Mitochondrial DNA-Restriction Fragment Length Polymorphism (RFLP), (v) Random Amplified Polymorphic DNA (RAPD) PCR analysis, (vi) Microsatellite analyses; (vii) Transposon-associated Delta Sequence Analysis, and (viii) Multilocus Sequence Typing (MLST) [5].

Molecular typing of the grape microbiome: the good, the bad and the ugly
The task of sorting the “desirable” from the unwanted, “spoiler” grape microflora, has been greatly simplified by the highly sensitive, Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (ITS-RFLP) assay [2, 6].

This assay takes advantage of the hypervariable Internal Transcribed Spacer (ITS) regions that are species-specific, and which play an essential role in the development of functional ribosomal RNA (rRNA). In yeasts, the ITS1 domain occupies the region between the 18S rRNA and the 5.8S rRNA genes, while the ITS2 region separates the 5.8S rRNA and the 28S rRNA genes [7]. In most bacterial genomes, the region separating the 16S and 23S rRNA genes contains a hypervariable ITS region. This domain is highly polymorphic and species-specific [8]. Thus, PCR amplification of ITS regions, combined with RFLP analysis of the digested amplicons, is a powerful tool to identify different species of grape microflora.

Concluding remarks
The use of molecular genetic techniques in the identification, manipulation and analysis of grape microflora, have added scientific rigor to the process. Furthermore, it is possible to create novel microbial strains and consistently-reproducible wine varieties that hitherto, did not exist.   

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REFERENCES
1.    PRETORIUS, IS. “Conducting wine symphonics with the aid of yeast genomics”.  Beverages 2016; 2: 36-63. doi: 10.3390/beverages2040036.
2.    APONTE, M, BLAIOTTA, G. “Potential role of yeast strains isolated from grapes in the production of Taurasi DOCG”. Front Microbiol 2016; 7: 809.
3.    JOLLY, NP, VARELA, C, PRETORIUS, IS. “Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered”. FEMS Yeast Res 2014; 14(2): 215-237.
4.    “Avoiding spoilage from lactic acid bacteria”. The Australian Wine Research Institute. Fact Sheet: Winemaking [Updated 2016].  https://www.awri.com.au/wp-content/uploads/2011/06/Avoiding-spoilage-from-LAB.pdf
5.    TOFALO, R, PERPETUINI, G, SCHIRONE, M, et al. “Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods”. Front Microbiol 2013; 4: 166
6.    BARATA, A, GONZÁLEZ, S, MALFEITO-FERREIRA, M, et al. “Sour rot-damaged grapes are sources of wine spoilage yeasts”. FEMS Yeast Res 2008; 8(7): 1008-1017.
7.    ESTEVE-ZARZOSO, B, BELLOCH, C, URUBURU, F, et al. “Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers”.  Int J Syst Bacteriol 1999; 49 Pt 1: 329-337.
8.    YAVUZ, E, GUNES, H, BULUT, C, et al. “RFLP of 16S-ITS rDNA region to differentiate Lactobacilli at species level”. World J Microbiol Biotechnol 2004; 20: 535-537.

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