**6. Combination of microbial evolution studies with metabolism analysis could provide indications of the microbial terroir**

The different varieties of grapevine (*Vitis vinifera* L.) are differentiated by a unique pool of compounds or chemical precursors that influence the aromatic composition of the produced wines. For instance, linalool is a typical characteristic aroma of Muscat varieties, while methoxypyrazine derivatives characterize the varieties Sauvignon blanc and Cabernet Sauvignon [64]. Apart from the grapevine variety, the degree of ripening, as well as the agronomic and oenological techniques applied, influence also wine's aromatic profile [65–71]. The metabolic reactions performed in wines, due to the specific enzymatic activity of selective wine yeasts that assist to the catabolism of sugar molecules and other ingredients, in order for the aroma compounds to be released have been reviewed extensively [72–74]. Indicatively, the basic yeast enzymes implicated in flavor compounds' secretion from the catabolism of grape components include: (a) glycosidases, such as α-l-arabinofuranosidase, α-l-rhamnosidase or β-d-apiosidase and β-d-glucosidase, which lead to the release of aromatic compounds found in the

**105**

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

bound aroma sections of diglycosides, glucosides and chemical compounds including terpene diols, terpenols, C13-norisoprenoids [72, 75, 76]. These enzymes

are produced mainly by the genera *Saccharomyces, Debaryomyces, Candida, Hanseniaspora/Kloeckera, Metschnikowia, Zygosaccharomyces Kluyveromyces, Pichia, Schizosaccharomyces and Saccharomycodes, Brettanomyces, Torulaspora* and *Trichosporon* [70, 77–91]. *(b) Carbon-sulfur lyases, that catalyze the release of volatile or varietal thiols from glutathionated thiol precursors produced by yeasts, including S. cerevisiae, Pichia kluyveri, Candida zemplinina, Metschnikowia pulcherrima*, *Hanseniaspora uvarum, Kluyveromyces thermotolerans and Torulaspora delbrueckii*

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 [92]. 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 [96–99]. 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 [57]. The majority of the fermentative aroma metabolites are characterized by elevated sensory thresholds [70]. 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 [95]. 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

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 [12] 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 [96] 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-*Saccharomyces* yeasts derived from three wine appellations in Spain and relating phylogenetic data with specific wine-associated enzymatic capabilities from glycosidases (β-glucosidase, α-Larabinofuranosidase and β-D-xylosidase), β-lyases, pectinases, proteases, cellulases and sulfite reductases, indicated distinct origin-associated clusters for species such as *A. pullulans*, *T. delbrueckii*, *W. anomalus*, *H. uvarum and L. thermotolerans*.

Importantly, genetic variations among microbial strains may alter the overall profile of the wine's volatiles, proposing the influence of another contributing factor

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

could provide a characteristic *terroir* to the region [57].

[92–94].

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

bound aroma sections of diglycosides, glucosides and chemical compounds including terpene diols, terpenols, C13-norisoprenoids [72, 75, 76]. These enzymes are produced mainly by the genera *Saccharomyces, Debaryomyces, Candida, Hanseniaspora/Kloeckera, Metschnikowia, Zygosaccharomyces Kluyveromyces, Pichia, Schizosaccharomyces and Saccharomycodes, Brettanomyces, Torulaspora* and *Trichosporon* [70, 77–91]. *(b) Carbon-sulfur lyases, that catalyze the release of volatile or varietal thiols from glutathionated thiol precursors produced by yeasts, including S. cerevisiae, Pichia kluyveri, Candida zemplinina, Metschnikowia pulcherrima*, *Hanseniaspora uvarum, Kluyveromyces thermotolerans and Torulaspora delbrueckii* [92–94].

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 [92]. 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 [96–99]. 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 [57]. The majority of the fermentative aroma metabolites are characterized by elevated sensory thresholds [70]. 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 [95]. 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 *terroir* to the region [57].

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 [12] 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 [96] 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-*Saccharomyces* yeasts derived from three wine appellations in Spain and relating phylogenetic data with specific wine-associated enzymatic capabilities from glycosidases (β-glucosidase, α-Larabinofuranosidase and β-D-xylosidase), β-lyases, pectinases, proteases, cellulases and sulfite reductases, indicated distinct origin-associated clusters for species such as *A. pullulans*, *T. delbrueckii*, *W. anomalus*, *H. uvarum and L. thermotolerans*.

Importantly, genetic variations among microbial strains may alter the overall profile of the wine's volatiles, proposing the influence of another contributing factor

*Advances in Grape and Wine Biotechnology*

appeared to be the dominant genus in all wine samples.

*osmophila* was also dominant.

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*

Metagenomic analysis, applied to reveal the spatial distribution of the microbial

**6. Combination of microbial evolution studies with metabolism analysis** 

The different varieties of grapevine (*Vitis vinifera* L.) are differentiated by a unique pool of compounds or chemical precursors that influence the aromatic composition of the produced wines. For instance, linalool is a typical characteristic aroma of Muscat varieties, while methoxypyrazine derivatives characterize the varieties Sauvignon blanc and Cabernet Sauvignon [64]. Apart from the grapevine variety, the degree of ripening, as well as the agronomic and oenological techniques applied, influence also wine's aromatic profile [65–71]. The metabolic reactions performed in wines, due to the specific enzymatic activity of selective wine yeasts that assist to the catabolism of sugar molecules and other ingredients, in order for the aroma compounds to be released have been reviewed extensively [72–74]. Indicatively, the basic yeast enzymes implicated in flavor compounds' secretion from the catabolism of grape components include: (a) glycosidases, such as α-l-arabinofuranosidase, α-l-rhamnosidase or β-d-apiosidase and β-d-glucosidase, which lead to the release of aromatic compounds found in the

**could provide indications of the microbial terroir**

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* 

**104**

to regional characteristic terroir. Genetic variances between *S. cerevisiae* strains lead to alterations in the wines' metabolic profile affecting their sensory qualities [100–105]. Fluctuations in the expression levels of key enzymes affecting wine's aroma among different *S. cerevisiae* strains isolated from diverse geographic areas of New Zealand indicated correlations among geographic region and genetic background as well as the phenotypic profile of *S. cerevisiae* [103]*.* However, the phenotypic plasticity of *S. cerevisiae* to produce altered phenotypes based on the fermentation microenvironment was found to affect the metabolic profile of wines [104].

Moreover, genotypic characterization of different strains of *O. oeni*, *isolated from diverse geographic regions* during the process of malolactic fermentation, *revealed a highly diverse genetic background among the strains derived from different locations*, *but also strains categorized in the same phylogenetic group* were detected in diverse regions, adapted in the same type of wine [105]. Noteworthy, the genomic, transcriptomic, and proteomic profile of various *O. oeni* strains was found to be strongly influenced by microenvironmental conditions during winemaking [106–108].

*Brettanomyces bruxellensis* (or *Dekkera bruxellensis*), a yeast implicated in wine spoilage producing volatile phenols that create unpleasant flavors, was found to be composed by strains with differences in their genetic background that affected their adaptation in the wine-producing environment [109–112]. Microsatellite analysis of 1488 *B. bruxellensis* strains isolated from diverse geographic locations identified that the *B. bruxellensis* population was differentiated not only based on ploidy level, culture method, and fermentation environment but also on the origin of isolation [112], highlighting again the influence of geographic region in combination with additional influencing factors to microbial terroir formation.

## **7. Conclusion**

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.
