**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 [12–16] (**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 [12]. 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

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**Figure 2.**

*Contribution of the Microbiome as a Tool for Estimating Wine's Fermentation Output…*

individual vineyards and the viticultural area, as revealed by random forest machine learning models [12]. Markedly, a negative association among the fermentation rate

*fermentation. (E) Representation of the relative abundance of fungal genera at the middle of fermentation.* 

*Spatial distribution of the microbial communities shaping from the initiation of fermentation until wine production regarding the studies of Stefanini et al. [16], Marzano et al. [14], Wei et al. [15], and Pinto et al. [13]. (A) Representation of the relative abundance of bacterial families at the beginning of fermentation. (B) Representation of the relative abundance of bacterial families at the middle of fermentation. (C) Representation of the relative abundance of bacterial families at the end of fermentation. (D) Representation of the relative abundance of fungal genera at the beginning of* 

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 [13]. 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

as well as bacterial richness with various taxa, such as *Lactobacillus* spp., *H. uvarum*, and *Gluconobacter*, was observed, indicative of the ability of some bacteria to prevent alcoholic fermentation, probably due to antagonism for available nutritional sources with the alcoholic fermentation fermenters, such as *S. cerevisiae*, while others, such as *Pseudomonas*, were positively correlated in both wines. The malolactic fermentation (MLF) conducted in Cabernet limits the bacterial biodiversity of wines to the presence of members of the family *Leuconostocaceae* (*Oenococcus oeni*), whereas the fungal biodiversity, as well as the microbial diversity of Chardonnay wines, remained enriched throughout fermentation and wine production, possibly responsible for the more distinct both regional and vineyard discriminations of Chardonnay wines compare to Cabernet Sauvignon wines.

*(F) Representation of the relative abundance of fungal genera at the end of fermentation.*

*DOI: http://dx.doi.org/10.5772/intechopen.85692*

*Contribution of the Microbiome as a Tool for Estimating Wine's Fermentation Output… DOI: http://dx.doi.org/10.5772/intechopen.85692*

#### **Figure 2.**

*Advances in Grape and Wine Biotechnology*

*Variables related to the microbiome formation.*

Generally, many of these variables (e.g., climatic conditions or cultivar) are interdependent and may be clustered into broad groups of effects **(Figure 1)**. The study of Bokulich and Mills [17] has shown that grape-associated microbial region is totally related with varietal, biogeographical, and climatic factors across multiscale viticultural zones. According to other study [20], the distribution of yeast species promotes significantly intra-vineyard spatial fluctuations. Continuously, the heterogeneity of grape samples harvested from single vineyards at the same stage of ripeness might be related, at least in part, to differing microbial communities in different sections of the vineyard. The biodiversity of yeast species in grapes is affected by numerous biotic and abiotic factors, as well as the interactions among the resident populations. However, more studies need to be performed in order to confidently elucidate the vineyard and grapevine phyllosphere microbiome.

**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 [12–16] (**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 [12]. 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

**102**

**Figure 1.**

*Spatial distribution of the microbial communities shaping from the initiation of fermentation until wine production regarding the studies of Stefanini et al. [16], Marzano et al. [14], Wei et al. [15], and Pinto et al. [13]. (A) Representation of the relative abundance of bacterial families at the beginning of fermentation. (B) Representation of the relative abundance of bacterial families at the middle of fermentation. (C) Representation of the relative abundance of bacterial families at the end of fermentation. (D) Representation of the relative abundance of fungal genera at the beginning of fermentation. (E) Representation of the relative abundance of fungal genera at the middle of fermentation. (F) Representation of the relative abundance of fungal genera at the end of fermentation.*

individual vineyards and the viticultural area, as revealed by random forest machine learning models [12]. Markedly, a negative association among the fermentation rate as well as bacterial richness with various taxa, such as *Lactobacillus* spp., *H. uvarum*, and *Gluconobacter*, was observed, indicative of the ability of some bacteria to prevent alcoholic fermentation, probably due to antagonism for available nutritional sources with the alcoholic fermentation fermenters, such as *S. cerevisiae*, while others, such as *Pseudomonas*, were positively correlated in both wines. The malolactic fermentation (MLF) conducted in Cabernet limits the bacterial biodiversity of wines to the presence of members of the family *Leuconostocaceae* (*Oenococcus oeni*), whereas the fungal biodiversity, as well as the microbial diversity of Chardonnay wines, remained enriched throughout fermentation and wine production, possibly responsible for the more distinct both regional and vineyard discriminations of Chardonnay wines compare to Cabernet Sauvignon wines.

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 [13]. 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 [14]. Although a common microbiome core was identified among the three wine varieties, comprised by the genera *Candidatus liberibacter*, *Gilliamella*, *Gluconobacter*, *Halomonas*, *Halospirulina*, *Komagataeibacter*, *Pseudomonas*, and *Shewanella*, each wine was discriminated by a unique taxonomic signature. During malolactic fermentation *Shewanella*, *Halomonas*, and *Oenococcus* became the dominant genera, whereas at the end of fermentation, *Oenococcus*, with the species *Oenococcus oeni*, became the abundant bacterium of the three wines' microbiome. Similarly, HTS analysis of Cabernet Sauvignon samples from three different winery regions in Xinjiang province, China, from Fukang area, identified a common core microbiome composed mostly by the fungal genera *Aureobasidium*, *Pleosporaceae*, *Cryptococcus*, and *Dothideales* and the bacterial genera *Pseudomonas*, *Acinetobacter*, *Kaistobacter*, *Arthrobacter*, and *Sphingomonas* in all grape and grape juice samples analyzed, even though the relative abundances of those genera were different [15]. However, following malolactic fermentation, the microbial biodiversity was gradually reduced and limited mostly to the fungal genera *Aspergillus*, *Penicillium*, and *Alternaria*, while the slowgrowing, necessary for malolactic fermentation, lactic acid bacterium *Oenococcus* appeared to be the dominant genus in all wine samples.

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" [16]. As a result of the spontaneous fermentation, the complex microbial diversity which composed the grapes' microbiome, including *Aureobasidium pullulans*, *Starmerella meliponinorum* MS 2010, *Penicillium polonicum*, *Pichia membranifaciens*, *Candida zemplinina*, *Penicillium bialowiezense*, and *Candida ethanolic*, was limited to some specific wine yeast species, which existed in limited relative abundance before fermentation, such as *Saccharomyces cerevisiae*, *Pichia membranifaciens*, and *Hanseniaspora osmophila*. Even though the must from the different wineries had significantly different mycobiome, the dominant presence of *Saccharomyces* at the end of fermentation was observed in all must tested, except from the Poli must, in which *Hanseniaspora osmophila* was also dominant.
