Wine is the alcoholic beverage which is the product of alcoholic fermentation, usually, of fresh grape must. Grape microbiome is the source of a vastly diverse pool of filamentous fungi, yeast, and bacteria, the combination of which plays a crucial role for the quality of the final product of any grape must fermentation. In recent times, the significance of this pool of microorganisms has been acknowledged by several studies analyzing the microbial ecology of grape berries of different geographical origins, cultural practices, grape varieties, and climatic conditions. Furthermore, the microbial evolution of must during fermentation process has been overstudied. The combination of the microbial evolution along with metabolic and sensorial characterizations of the produced wines could lead to the suggestion of the microbial terroir. These aspects are today leading to open a new horizon for products such as wines, especially in the case of PDO-PGI products. The aims of this review is to describe (a) how the microbiome communities are dynamically differentiated during the process of fermentation from grape to ready-to-drink wine, in order to finalize each wine’s unique sensorial characteristics, and (b) whether the microbiome could be used as a fingerprinting tool for geographical indication, based on high-throughput sequencing (HTS) technologies. Nowadays, it has been strongly indicated that microbiome analysis of grapes and fermenting musts using next-generation sequencing (NGS) could open a new horizon for wine, in the case of protected designation of origin (PDO) and protected geographical indication (PGI) determination.
- next-generation sequencing
Fermented products are generated as a result of metabolic activities conducted by functional microbes, leading to the biochemical and organoleptic modification of the substrates in order to meet the requirements of the consumers . The dynamic interaction between the members of the microbial communities guiding the process of fermentation has great influence in the nutritional, hygienic, safety, and organoleptic characteristics of the final product . In a large number of fermented products, the formation of microbial biodiversity existing in the initial substrate is affected by a large number of factors, including the geographic origin, the cultural practices, differences among varieties, or the climatic conditions . The contribution of the microbial community configuration, which is governed by spatial factors, land topography, environmental factors, etc. that sustain the spatial structure of the inhabitants, and their potential relation with the metabolic and sensorial characterizations of the final product, has been under deep research, leading to the suggestion of the microbial terroir . The perspective of analyzing the microbial communities’ dynamics as progressively differentiated during the process of fermentation for the determination of microbial terroir has been applied in grapes and consequently its final fermented product, the wine [5, 6].
Traditionally winemaking process relies on spontaneous fermentation, which is conducted without the addition of chemical compounds or supplementary microbes at the beginning of the fermentation process. Under spontaneous fermentation conditions, the microbial community participating in fermentation and which is responsible for the quality of the final product is considered to be quite unpredictable. At the initial stages of fermentation, the microbial communities are comprised by a rich biodiversity of several yeast and mold species, including
The development of high-throughput sequencing technologies has allowed the evaluation of the microbial consortium comprising grapes’ microbiome in terms of revealing the concept of the microbial terroir [12, 13, 14, 15, 16]. The contribution of origin-associated factors of grape varieties, including climate and microclimate, region site, as well as grape cultivar, in the microbial community formation and the final metabolic profiles, has been recently investigated [12, 17, 18, 19, 20]. These studies have led to an improved spatial and temporal determination of the wine grapes’ microbiome and brought new insights into its dynamics and biodiversity, revealing a new horizon for the better characterization of this product, especially in the case of PDO and PGI wines’ designation. These labels were established by the European Union (EU) to guarantee the authentication of the local products produced in distinct geographic origin, applying traditional specialties. Metagenomic studies have been recently applied to identify the microbial communities that influence the original sensorial characteristics of PDO wines [14, 16].
The aim of this chapter is to extensively review all latest literature in the scope to investigate (a) how the microbiome communities are dynamically differentiated from grape to ready-to-drink wine, in order to finalize each wine’s unique sensorial characteristics ,and (b) whether the microbiome could be used as a fingerprint tool for regional characterization, based on high-throughput sequencing (HTS) technologies.
2. Methods to identify grape microbial species
Grapes are comprised by a complex microbiome, the members of which share different physiological characteristics and effects upon wine production. Some of them are present only in grapes and soil, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow during wine fermentation, constituting the wine microbial consortium. Several studies over the last years have reported that the biodiversity and the quantity of the microorganisms present on the surface of the grape berry are highly dependent on many factors, including the health state of the grapes, the temperature, the microclimate conditions, and the pesticide treatments [21, 22, 23]. Recently, the “terroir” idea was proposed to be extended to the microbiological aspect, indicating that the geographical distribution of the grape and soil microbiota is not randomly dispersed but is dependent on the cultivar, the location of the vineyard, and the vintage .
The application of culture-dependent methods is considered weak to support the terroir perspective, since less than 1% of the total population can be detected , and these methods also fail to detect viable but non-culturable organisms [25, 26, 27]. Additionally, the stressful environment shaped during winemaking due to the addition of SO2, high ethanol concentration, etc. forces a number of bacteria and yeast to enter a viable but non-culturable state (VBNC) [28, 29]. Even though still viable and maintaining a detectable metabolic activity, the microbial cells are unable to grow on culture media during VBNC status . Examples of such microorganisms include
The introduction of next-generation sequencing (NGS) technologies has significantly enhanced the information elicited from microbiological studies, allowing the distinction of the high-abundant species from the low-abundant, with detection sensitivities greatly higher than the previously used molecular techniques . For instance, analysis of the microbial communities’ formation existing on grape and during Carignan and Grenache must fermentation from three vineyards in Priorat (Spain) highlighted the ability of NGS to detect an increased amount of species compared to DGGE . Undoubtedly, NGS provides a new powerful tool, with elevated capabilities to enhance the understanding of the complexities of microbial communities as dynamically differentiated from grapes and its close environment to ready-to-drink fermented wine, in terms of diagnostic, monitoring, and traceability [16, 21, 35, 36, 37, 38]. Understanding the progressive alterations of the microbial diversity during fermentation using HTS technologies is considered a promising approach to reveal correlations between microbiomes and geographical origin.
3. Identification of the microbial communities
Overall, the fungal population at a phylum level is very similar and mainly comprised by
High-throughput sequencing studies have been applied to evaluate the bacterial communities associated with the vineyard. The most frequently detected phyla in vineyard soils and grapevine roots include
4. Factors affecting the microbial communities’ formation
Grapevines’ associated microbial communities originated from distinct geographic regions exhibit different profiles [13, 18, 34, 36, 55]. Each region is differentiated by the dominance of a few species per region. Indicatively,
Many studies suggested that yeast diversity is dependent on climatic and microclimatic conditions. Higher yeast diversity has been described for vintages with high rainfall [40, 57] probably due to substantial fungal proliferation. Dry wines are produced by grapes submitted to prolonged withering in order to become moderately dried. The climate, as well as the extent of the withering period, was found to affect the formation of the fungal microbiome on grape skins in
Vineyard factors such as grape variety and berry chemical components are often described to influence microbial diversity [11, 43, 61, 62]. For instance, in similar soil and climatic conditions,
The health status of berries can also affect the diversity of yeasts. The ascomycete
One of the factors found to contribute to microbial communities’ formation is the amount of SO2. Comparison of the bacterial community dynamics following the fermentation process of hand-harvested organically grown Riesling grapes following organic and conventional
Generally, many of these variables (e.g., climatic conditions or cultivar) are interdependent and may be clustered into broad groups of effects
5. Microbial evolution of must during spontaneous fermentation process
High-throughput sequencing techniques have allowed the discrimination of the microbial diversity as dynamically formed from the initiation of fermentation until wine production, identifying also the non-culturable microorganisms, as well as the limited represented species [12, 13, 14, 15, 16] (Figure 2). During the process of fermentation, the microbial community is reshaped and become dominated by the fermentative organisms. These alterations, however, are to a large extent dependent from the origin of the must/wine, including the winery and the grape variety . Metagenomic analysis of the microbial communities’ structure fluctuations formed throughout the fermentation of grapes obtained from American Viticultural Areas (AVA), for Cabernet and Chardonnay wines production, combined with metabolomic analysis, indicated that the characteristic microbial signatures of grapes and soil disappeared during fermentation to become replaced by characteristic fermentative microbes, but still, the microbial and wine metabolite profiles were able to distinguish the individual vineyards and the viticultural area, as revealed by random forest machine learning models . Markedly, a negative association among the fermentation rate as well as bacterial richness with various taxa, such as
In order to understand the association among the biogeographic distribution of wineries and wine microbiome of six different Portuguese wine appellations, HTS analysis was applied to reveal the dynamics of microbial communities’ formation following the different stages of spontaneous wine fermentations . The presence of an increased average microbial biodiversity dissimilarity among the grape microbiome from the different wine appellations (60.16 and 57.36% for eukaryotes and prokaryotes, respectively) indicated the elevated contribution of the vineyard environment in microbial communities’ shaping and consequently the influence of the initial microbiome to the uniqueness of the different appellation-derived wines. During the process of fermentation, the average microbial dissimilarity was reduced, due to alterations in the microbial biodiversity and dominance of specific, able to perform fermentation species, leading to the loss of the biogeographic profile, but still each wine was distinguished by its unique pattern of microbial biodiversity.
The high detection sensitivities of HTS technologies have allowed the identification of the rich bacterial biodiversity implicated in Cabernet, Negroamaro, and Primitivo Apulian red wines’ production process, highlighting the alterations in the bacterial population during vinification . Although a common microbiome core was identified among the three wine varieties, comprised by the genera
Metagenomic analysis, applied to reveal the spatial distribution of the microbial communities shaped in Vino Santo Trentino sweet wine, produced by Nosiola grapes from three wineries (Poli, Pedrotti, and Pisoni in the Italian Alps), indicated that a winery-specific “microbial-terroir” contributed mostly to the wines’ microbial community shaping, rather than a regional “terroir” . As a result of the spontaneous fermentation, the complex microbial diversity which composed the grapes’ microbiome, including
6. Combination of microbial evolution studies with metabolism analysis could provide indications of the microbial terroir
The different varieties of grapevine (
A great influence on the pool of the VOCs released in wine is due to the metabolic activities performed mostly by predominant yeasts, leading to secondary metabolites’ production during fermentation . These secondary aroma compounds include ethanol, CO2, and glycerol, as well as volatile fatty acids, such as acetic acid and propanoic and butanoic acid esters, higher alcohols and aldehydes, and volatile derivatives of fatty acids and nitrogen- and sulfur-comprising compounds, which have greater contribution to the secondary aroma profile [96, 97, 98, 99]. The spontaneous fermentation is conducted by autochthonous yeasts, which exist naturally on the surface of grapes. Increased biodiversity of yeast strains leads to elevated content of VOCs in wine . The majority of the fermentative aroma metabolites are characterized by elevated sensory thresholds . As a result, their combination shapes the characteristic aroma of wines. Importantly, some metabolic reactions performed by must microbiota are considered undesirable, since they spoil the quality of wine, such as by the acetic acid production . Botrytized wine fermentations were found to contain increased abundance of acetic acid bacteria (AAB) in comparison with unaffected wines [36, 64]. Based on that, the selective microbial communities which are related to specific grape varieties, originated from particular locations, may extract distinctive metabolites, the combination of which could provide a characteristic
The understanding of the contribution of the microbial communities in the sensorial characteristics of the wine requires the combination of metagenomic studies that will allow the identification of the wine’s microbiome, with transcriptomics or metabolomics, which will reveal the volatile profile of the produced metabolites. Bokulich and colleagues  proposed that by identifying the microbial pool which composes grapes, and based on the existed knowledge, a great amount of the produced in the wine metabolites could be predicted. Indeed, by applying metabolomics and associating them with microbial communities—metagenomics—they discovered marker metabolites able to differentiate AVAs. Additionally, through a statistical model, they suggested that the grape must microbial conformation is able to predict the metabolites comprising the produced wine, proposing that regional microbial composition patterns may be able to characterize the wine physiognomies. Similarly, Belda and co-workers  suggested that the enzymatic activities of the wine-related microbial species population may predict the influence of the produced metabolites on wine aroma and establish region-derived clusters, via combination of metagenomics with information extracted by species-related enzymatic profiles analysis. Through gathering numerous non-
Importantly, genetic variations among microbial strains may alter the overall profile of the wine’s volatiles, proposing the influence of another contributing factor to regional characteristic terroir. Genetic variances between
Moreover, genotypic characterization of different strains of
Regional characteristics such as climate, agronomic practices, grape variety, and soil chemistry may influence the composition of the local microbial communities creating a characteristic regional microbial profile described with the term “microbial terroir.” The composition of a particular variety grape microbiome, beyond its dynamic fluctuations during fermentation, was found to be able to provide indications regarding the chemical composition and the sensorial characteristics of the produced wines. The existence of specific regional microbial biomarkers, able to predict the metabolic composition of the wine, is a powerful indication of the existence of a clear association between region and local microbiome. Future studies based on the combination of HTS technologies with metabolomic studies may provide more enhanced evidence regarding the contribution of the regional microbial communities to wines’ sensorial characteristics.
The authors would like to acknowledge the financial contribution from project AGROID funded by the INTERREG Greece-Cyprus 2014–2020 Program which is co-funded by the European Union (ERDF) and National Resources of Greece and Cyprus.
Conflict of interest
None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias this publication.