The microbial ecology of wine grape berries

Abstract

Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing.

The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect.

Introduction

Grapes used for making wine have been studied for several decades. The earlier work of Louis de Pasteur, by the last quarter of the XIXth century, already showed that the microorganisms responsible for wine fermentations are yeasts present on the grapes. Since then a huge amount of information has been gathered on yeast dissemination in wine associated environments but ecological relationships are still to be fully understood. The prime focus has been the study of Saccharomyces cerevisiae given its relevance to winemaking. Earlier isolations using grape juice fermentations lead to the conclusion that this species was frequent in grapes (as reviewed by Vaughan-Martini and Martini, 1995). However, the use of direct isolation techniques showed a much different picture, revealing that Saccharomyces spp. is an absent or rare contaminant of grapes (Davenport, 1973, Davenport, 1974). This fact has lead to a still lively debate on the origin of the yeasts responsible for wine fermentation, being hypothesised that S. cerevisiae may be regarded as the first domesticated microbe (Martini, 1993).

Pioneering works on grape microbial ecology describe the main yeast species isolated from grapes and their environs (as reviewed by Fleet et al., 2002), while bacteria have been less studied. The recent increase in the number of grape-related yeast ecological studies did not contribute to a significant body of new knowledge. On the other hand, other areas of yeast ecology, like those related with plants, insects, soil or extreme habitats, have witnessed a significant development (Rosa and Péter, 2006) which provide useful guidance to study grape microbiota. In fact, most recent grape-related works are mostly dissemination studies. Microbial ecology is a wider concept, studying the ecosystems with their microbial interactions, microbial vectors, sources and sinks of microorganisms (Boddy and Wimpenny, 1992). Herman Phaff, the pioneer of yeast ecology, described ecology as “where microbes live and why they live in one habitat and how yeasts interact with other microorganisms” (Lachance, 2003). This global approach has been scarcely adopted to study the vineyard environment (Fleet et al., 2002). There are large gaps in the knowledge of the diversity and significance of yeast–yeast, yeast–fungi and yeast–bacteria interactions on grapes (Fleet, 2003). This may be because the microorganisms relevant to vine and grapes diseases (phytopathogenic parasitic and saprophytic fungi) do not survive and grow in wine, while the species of the wine microbial consortium (WMC: yeasts, acetic acid bacteria and lactic acid bacteria) are not responsible for those diseases. The apparent rising of sour rot incidence, where the agents are part of the WMC (Barata et al., 2008a, Barata et al., in press), justify a reappraisal of the microbial ecology of grapes.

In this review we will attempt to make an update of the current awareness on the microbial ecology of grape surfaces, describing the species diversity, the factors affecting the species balance and the interactions with other microorganisms and vectors. We will also evaluate the current methods for microbial recovery and identification, trying to explain the contradictory results often found in literature. Special attention will be paid to the distribution of WMC species responsible for wine spoilage and to the effect of grape diseases on wine quality, given their technological importance. The question of the origin and dissemination of the WMC in the environment will be discussed aiming to provide future research directions.

The microbial species recovered from grapes may be divided into several groups according to their technological significance in grape and wine production. Under this view, microorganisms are characterised as a function of their effect on grape and wine quality.

The vine plant and grapes may be affected by a series of diseases of which the most well known are downy mildew (Plamospara viticola), powdery mildew (Erysiphe necator) and grey rot (Botrytis cinerea), which are mostly prevented by phytochemical application. In addition, grapes may also bear saprophytic moulds (e.g. Cladosporium spp., Aspergillus spp., Penicillium spp.) responsible for several grape rots or mycotoxin production. However, these fungi do not have the ability to grow in wines and their effect on wine quality is due to grape damage. Contrarily, the microorganisms of the WMC are able to survive or grow on wine, depending on the efficiency of adequate processing measures. Thus, based on the concept already suggested by the authors (Loureiro and Malfeito-Ferreira, 2003, Malfeito-Ferreira, 2011), the species in this consortium may be grouped into: (i) easily controllable or innocent species, without the ability to spoil wine when good manufacturing practises (GMP's) are applied; (ii) fermenting species, responsible for sugar and malic acid conversion; and (iii) spoilage sensu stricto species responsible for wine alteration even when GMP's are believed to be applied. Table 1 lists the most relevant microorganisms of referred groups, including recognised empirical denominations.

Concerning yeasts, the innocent group includes basidiomycetous species which are regarded as irrelevant to winemaking due to their inability to ferment juice sugars or to survive in wines. The ascomycetous dimorphic fungus, Aureobasidium pullulans (also called black yeast), a common yeast-like species, is also technologically irrelevant. The oxidative, weakly fermentative or fermentative ascomycetous species (Candida spp., Kloeckera apiculata/Hanseniaspora uvarum, Metschnikowia spp., Pichia spp.) are present in pre-fermentation steps or at the beginning of fermentation. The fermentation ability is not a well defined taxonomic feature and several species may be regarded as weakly fermentative or not. Among these, apiculate yeasts are determined by their microscopical shape and some strains may produce off-flavours in juices before or during fermentation (Romano, 2002). Film-forming yeasts (e. g. Pichia) owe their denomination to the ability of forming pellicles on the surface of bulk wines, being common contaminants of grapes, juices and wines, with the ability to produce off-flavours. Both apiculate and film-forming yeasts are regarded as contaminants because good manufacturing practises prevent their activity. Fermentative yeasts include those responsible for wine fermentation, where S. cerevisiae is the most important, but other species (S. bayanus, S. pastorianus and S. paradoxus) may also conduct or participate in the process (Skelin et al., 2008, Arroyo-López et al.,, 2010). These species may also be seen as wine spoilers if their activity persists beyond the fermentative steps of wine or sparkling wine production. The exception is that of Saccharomyces spp. forming desirable films on the surface of particular oxidative ageing processes of Sherry-like wines (Farris et al., 2002). Additionally, fermentative species comprise the spoilage sensu stricto yeasts which are technologically relevant due to their ability to spoil wines, either by off-flavour production (e. g. Dekkera bruxellensis) or sediment and cloudiness formation (e. g. Zygosaccharomyces bailii), under conditions following the GMP rules.

Concerning bacterial species, acetic acid bacteria are regarded as innocent because they are easily controllable by GMP's in the winery, although the exaggerated production of acetic acid during grape sour rot is a serious threat to wine quality. The physiological diversity of lactic acid bacteria does not allow a precise assessment of their technological significance. The typical agent of malolactic fermentation is O. oeni while Lactobacillus spp. and Pediococcus spp. may be responsible for spontaneous fermentations (Lerm et al., 2010). These species may spoil wine when their activity goes beyond malic conversion, particularly in high pH wines, producing off-flavours or biogenic amines (Arena et al., 2011, Capozzi et al., 2011, Pan et al., 2011). Grapes also bear a wide diversity of bacterial species common in nature or in other food related environments (Table 1). However, they should not be integrated in the WMC because they do not influence wine quality.