Changes in sour rotten grape berry microbiota during ripening and wine fermentation
Highlights
► Grape damage is the main driving force altering berry microbiota. ► Rotting process induced deep changes on yeasts and acetic acid bacteria populations. ► Zygoascus hellenicus and Issatchenkia spp. may be regarded as markers of sour rot. ► Acetobacter orleaniensis and A. syzygii were only recovered from sour rotten grapes.
Introduction
Grape quality may be affected by a wide range of rots. Among them, sour rot is an emergent grapevine disease affecting late ripening cultivars with tightly-packed, thin-skinned and dense bunches close to harvesting, causing heavy crop losses and being detrimental to juice and wine quality (Bisiach et al., 1986, Wolf et al., 1990, Zoecklein et al., 1992). Rotten bunches have a strong and pungent odour of vinegar as a result of the production of high levels of acetic acid, accompanied by high concentrations of glycerol, ethyl acetate, ethanol, acetaldehyde and galacturonic and gluconic acids (Marchetti et al., 1984, Zoecklein et al., 2001). These products are the result of a mixed population of yeasts and acetic acid bacteria (AAB) (Acetobacter spp. and Gluconobacter spp.) (Bisiach et al., 1986, Blancard et al., 2000, Gravot et al., 2001).
Berry rupture is associated with a sudden increase in yeast load up to about 106–108 CFU/g and deep alterations in species diversity occur when compared with sound grapes (Barata et al., 2008a, Barata et al., 2008b). The main yeast species recovered from sour rotten grapes are Candida krusei, Issatchenkia orientalis, Kloeckera apiculata/Hanseniaspora uvarum, Saccharomycopsis vini, Candida steatolytica (syn. Zygoascus hellenicus), Torulaspora delbrueckii, Issatchenkia terricola and Zygosaccharomyces bailii (Barata et al., 2008a, Barata et al., 2008b, Bisiach et al., 1982, Bisiach et al., 1986, Guerzoni and Marchetti, 1987, Guerzoni and Marchetti, 1982, Marchetti et al., 1984). The population size of AAB on healthy grapes is typically low (102–103 cells/g) and Gluconobacter oxydans is the species most represented, while in grapes damaged by Botrytis cinerea (grey rot), AAB populations can reach up to 105–106 cells/g, comprising Gluconobacter spp. and Acetobacter spp., mainly A. aceti and A. pasteurianus (Barbe et al., 2001). Undamaged grapes contains low populations of LAB, not exceeding 103 CFU/g and the initial titer in must is low (Bae et al., 2006, Fugelsang, 1997, Lafon-Lafourcade et al., 1983). Only a few LAB species of the genera Lactobacillus spp. (Lb.), Leuconostoc spp. (Lc.), Pediococcus spp. (P.), Oenococcus spp. (O.) and Weissella spp. (W.) can grow in must and wine (König and Fröhlich, 2009). The species Oenococcus oeni, regarded as the main agent of malolactic fermentation (Henick-Kling, 1993, Lonvaud-Funel, 1995, Lonvaud-Funel, 1999) has been seldom isolated from grapes in the vineyard (Renouf et al., 2007). Several other bacterial species have also been isolated from sound and grey rotten grapes (Nisiotou et al., 2011). However, these species have no technological significance in winemaking, being probably only a result of environmental contamination of berry surfaces.
Previous work from our laboratory, based on accurate berry sampling, has shown that grape damage by sour rot induced dramatic increases on yeast counts and number of species since the beginning of grape ripening (Barata et al., 2008a, Barata et al., 2008b). However, the knowledge of the complex microbial changes that lead to sour rot disease has not yet been completed and clearly understood, particularly in regard to the role and evolution of bacterial populations. Therefore, the purposes of this work were (i) to investigate the changes of the overall grape contaminants including yeasts, AAB and LAB populations present on healthy and sour rotten berries surfaces during different stages of ripening, and (ii) to study the fate of these populations during winemaking at winery level. For that, a careful berry sampling followed by isolation and enumeration on both general purpose media and selective and differential media, together with identification by molecular methods, were performed.
Section snippets
Grape samples
During the 2007 vintage, healthy and sour rot affected bunches of Vitis vinifera L. cv. Trincadeira red grape variety were selected from a experimental vineyard of Instituto Superior de Agronomia, located in Tapada da Ajuda, Lisbon, Portugal (latitude 38° 42′31.57″ N and longitude 9° 11′14.01″ W). Just after the veraison period, the Trincadeira parcels were visually inspected and vines containing bunches with sour rot symptoms were marked. Two sets of three different vines (duplicates) were
Isolation and identification of yeasts, AAB and LAB
A total of 203 yeasts isolates, recovered from sound and rotten grape samples during the 3 ripening phases, and 97 yeast isolates obtained from grape musts and wine samples throughout the 3 winemaking phases, were selected for identification. A total of 15 species were successfully identified by comparison of the CfoI, HaeIII and HinfI restriction profiles contained in the Yeast-id database (see Table S1 in supplementary material). The DraI endonuclease enabled the identification of C.
Conclusions
The results obtained in this work support our view that grape damage is the main driving force altering berry microbiota. Grape damage increases sugar accessibility and creates opportunities for new species to become established. Moreover, we believe that many of the apparently contradictory results found on literature regarding the factors affecting yeast diversity on grapes, may be explained by using grape bunches without separating damaged berries. Undoubtedly, deep changes on the yeast
Acknowledgements
This work was partially funded by the Portuguese Science and Technology Foundation (FCT) and by POCI 2010, participated by the European fund FEDER under the projects POCI/AGR/56771/2004 and PTDC/AGR-ALI/101393/2008. A. Barata was the recipient of a PhD grant (Ref. SFRH/BD/28451/2006) from the FCT.
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