Changes in sour rotten grape berry microbiota during ripening and wine fermentation

Abstract

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes.

Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.

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.