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Molecular Identification of Bacteria in Slovak Small Carpathian Wine Region

Explore the bacterial diversity in grapes and must from the Small Carpathian wine region in Slovakia using culture-based and culture-independent methods. Gain insights into the impact of bacteria on wine quality.

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Molecular Identification of Bacteria in Slovak Small Carpathian Wine Region

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  1. Molecular identification of bacteria on grapes and in must from Small Carpathian wine-producing region (Slovakia) T. Kuchta1, D. Pangallo2, Z. Godálová1, A. Puškárová2, M. Bučková2, K. Ženišová1, L. Kraková2 1Food Research Institute, National Agricultural and Food Centre, Bratislava, Slovakia 2Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia

  2. Slovakia

  3. Small CarpathianWine-producing region

  4. Small CarpathianWine-producing region

  5. Small Carpathians

  6. Twotypical grape varieties: • Blaufränkisch • Grüner Veltliner • Modra, vintage 2013 Samples:- grapes, wash (G)- initial phase must (M1)- middle fermenting must, 7-10 days (M2)- must at the end of fermentation, almost wine (M3)

  7. Besides the quality of grapes,microflora determines the quality of wine: * yeasts are responsibleforprimaryfermentation ** bacteriainfluencethequalityofwine • in a positiveway (malolacticfermentation, formation/transformationofvolatilearoma-activecompounds) • or in a negativeway (formationofaceticacid, biogenicamines)

  8. Culture-based approach for bacteria: Decimaldilutionscultured on electivemedia(supplementedwithcycloheximide): • R2A • GYC (forcultivationofaceticacidbacteria) • MRS (forcultivationoflacticacidbacteria) • MRS-T (suppl. withtomatojuice, cysteine and malicacid; for Oenococcusspp.) Colonies DNA  f-ITS PCR  capillary electrophoresis Selectedcolonies 16S rDNAsequences BLAST

  9. Culture-independent approach for bacteria: • DNA extraction (DNeasy, Qiagen) • semi-nestedPCRfor 16S rDNAamplification • cloning in pGEM-TEasyvector (Promega) • DGGE • cloneswithdifferentDGGEcharacteristicssequenced and identified by BLAST in GenBank

  10. Culture-based quantitative data on bacteriaisolated on different agar media during the production of wine - decrease in bacterial isolates in M3

  11. Culture-based identification of bacteria in wine • large diversity (16 genera) detected on R2A medium (dominant: Bacillus and Gluconobacter) • Leuconostoc, Gluconobacter and Acetobacter preferentially isolated on GYC medium • Lactobacillus, Leuconostoc and Gluconobacter isolated on MRS and MRS-T • larger diversity recovered on MRS than on MRS-T

  12. Clustering of isolates by f-ITS • Each f-ITS profile contained 1 – 5 peaks • 488 isolates were grouped into 45 clusters • Bacillus was heterogenous – 36 isolates in 7 clusters • Gluconobacter – 75 isolates in 4 clusters • Lactobacillus – 161 in 5 clusters

  13. Comparison of the bacterial diversityondifferent cultivation media

  14. Bacterial species isolated on different media in various wine production stages of Blaufränkisch

  15. Bacterial species isolated on different media in various wine production stages ofGrüner Veltliner

  16. Identification of bacterial isolates

  17. Identification of bacterial isolates (continued)

  18. Identification of bacterial isolates (continued) Members of the same genus continuously identified in different vinification stages are described within grey squares.

  19. Culture-independent analysis • Blaufränkisch – 27 bacterialgenera and 1 cyanobacterial • Grüner Veltliner – 17 bacterial genera Bacterial classes:

  20. Culture-independentanalysis • Blaufränkisch – 27 bacterialgenera and 1 cyanobacterial • Grüner Veltliner – 17 bacterial genera • The 16SrRNA sequences of bacterial clones were deposited in the GenBank database under the accession numbers KR704489 - KR704568. • Fromdifferentwineproductionsteps, chloroplast 16S rDNA and mitochondrial 18S rDNAofVitisviniferaweredetected

  21. Culture-independent identification of bacteria

  22. Culture-independent identification of bacteria (cont.)

  23. Culture-independent identification of bacteria (cont.) Members of the same genus continuously identified in different vinification stages are described within grey squares.

  24. - Detailed data on bacterial microflora of two typical wines of the Small Carpathian wine-producing regions were obtained- Data and isolates provide the basis for improvement of the quality of regional wines- The two scientific approaches produced complementary information on bacterial diversity

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