**1. Introduction**

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 [1]. 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 [2]. 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 [3]. 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 [4]. 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 *Metschnikowia*, *Candida*, *Hanseniaspora*, *Pichia*, *Lachancea*, *Kluyveromyces*, and *Saccharomyces* [7–9]*.* During must fermentation, the alcoholic fermentation conducted elevates the ethanol content and establishes the basic fermenters, such as *Saccharomyces cerevisiae,* among the predominant species [9]. Their dominant presence during the fermentation process has led to the isolation of several *S. cerevisiae* strains, which have been extensively studied for the potential application of their technological characteristics [9–11].

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 [12–16]. The contribution of originassociated 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, 17–20]. 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

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

the cultivar, the location of the vineyard, and the vintage [17].

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 [21–23]. 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 application of culture-dependent methods is considered weak to support the terroir perspective, since less than 1% of the total population can be detected [24], and these methods also fail to detect viable but non-culturable organisms [25–27]. 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 [30]. Examples of such microorganisms include *Candida stellata*, *Brettanomyces bruxellensis*, *S. cerevisiae*, and *Zygosaccharomyces bailii* [27]. In order to study the existence of bacteria during VBNC, microbiologists have applied alternative culture-independent techniques. Three of the main culture-independent techniques applied include quantitative real-time PCR (qPCR), restriction fragment length polymorphism (RFLP), and denaturing gradient gel electrophoresis (DGGE) [24, 27, 31–33]. Still, the detection sensitivities of these techniques remain limited due to the predominance of certain yeast such as *C. zemplinina* and *S. cerevisiae* during fermentation, which restrict the

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 [24]. 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 [34]. 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, 35–38]. 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.

*Terroir* is characterized by a multi-complex ecosystem where the vine (genetic material and cultural practices) interacts with the environmental factors (i.e., soil, climate, microclimate, humans, etc.) affecting the quality and typicity of the wine produced in a particular location. The understanding of the microbial terroir involves the identification of the microbes shaping grapes' environmental communities and the evaluation of their diversity dynamical evolution throughout the different stages of fermentation, until wine production. During natural fermentation the complex microbial communities that comprise the grape microbiome, including, yeasts, yeast-like fungi, and bacteria, are under the selective pressure of the

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

detection of low-abundant species.

**3. Identification of the microbial communities**

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

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 [21–23]. 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 [17].

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 [24], and these methods also fail to detect viable but non-culturable organisms [25–27]. 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 [30]. Examples of such microorganisms include *Candida stellata*, *Brettanomyces bruxellensis*, *S. cerevisiae*, and *Zygosaccharomyces bailii* [27]. In order to study the existence of bacteria during VBNC, microbiologists have applied alternative culture-independent techniques. Three of the main culture-independent techniques applied include quantitative real-time PCR (qPCR), restriction fragment length polymorphism (RFLP), and denaturing gradient gel electrophoresis (DGGE) [24, 27, 31–33]. Still, the detection sensitivities of these techniques remain limited due to the predominance of certain yeast such as *C. zemplinina* and *S. cerevisiae* during fermentation, which restrict the detection of low-abundant species.

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 [24]. 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 [34]. 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, 35–38]. 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.
