Identification of non-
Wine is an ancient and popular alcoholic beverage made from fermented grapes. Different yeasts and bacteria strains produce different styles of wines. Over time, the inoculation of Saccharomyces cerevisiae strains to produce wine has been the common practice in the wine industry, and the other species of yeasts have been considered undesirable for the alcoholic fermentation. However, in the last decades, the use of wild or indigenous yeasts and lactic acid bacteria strains has significantly increased. Wild yeasts and lactic acid bacteria are interesting microorganisms that contribute to differentiate the wine character of a region. The production of wines by spontaneous or inoculated fermentations by selected wild microorganisms may be an interesting tool to improve the quality of wines. This chapter summarizes relevant aspects of these microorganisms related to this scientific field.
Wine is an ancient and popular alcoholic beverage made from fermented grapes. The wine quality is determined by many factors, including the climate, the soil characteristics, the grape variety and the production processes, such as the viticultural practices, the winemaking techniques and the aging period. Among these factors, the fermentations carried out during winemaking, mainly alcoholic and malolactic fermentation, strongly influence on the wine composition. Different yeasts and bacteria strains produce different styles of wines derived from the biotransformation involved in both fermentations. Generally, the alcoholic fermentation (AF) is conducted by yeasts which convert sugars into ethanol, carbon dioxide and other minor metabolites. On the other hand, the malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB), which mainly convert malic acid into lactic acid and carbon dioxide. These metabolic processes are complex and sophisticated and sometimes may induce undesirable metabolite production pathways. Accordingly, an adequate selection of the yeast and bacteria strains is an important task for winemakers.
Over time, the inoculation of
2. Wild microorganisms associated to wine fermentations
The biotransformation of grape juice into wine is a complex ecological and biochemical process involving the sequential development of several microbial species such as yeasts, bacteria and fungi. Yeasts are the most important microorganisms involved in this process, being
LAB are the second important group of wine microorganisms, which are also present in grapes. The LAB of wines, musts and grapes belong to the genera
3. Population dynamics
The main important microorganisms present in grapes are yeasts and in a minor proportion LAB, acetic acid bacteria and fungi. The content and diversity strongly depend on the sanitary status of grapes. Although grape musts are relatively complete in nutrients, its low pH and its high sugar content convert them in a selective media in which only a few bacteria and yeasts species can grow. The number of yeasts on the grape berry just before harvest varies from 103 to 106 cells/mL depending on the abovementioned factors . The predominant wild species on the surface of grape berries are
Regarding LAB, the population and behavior mainly depend on the pH and the SO2 content, and they can reach 102–104 cells/mL populations after grape crushing. In general, an increase on the pH involves higher LAB populations and diversity. At this initial fermentation stage, the four genera abovedescribed can be commonly identified, although the greatest diversity of LAB species is mainly detected during the AF. During the first days of the AF, the LAB population generally increases to a maximum of 104 cells/mL and then decreases until 102 cells/mL. At the end of the AF,
4. Spontaneous and inoculated fermentations
There are a lot of different species in grapes that can participate on the wine fermentations. In general, the AF is conducted by a mixture of yeasts species . The AF can be conducted spontaneously without inoculating any yeast strains or by the inoculation of the specific strains, commercial or wild. The most common worldwide practice is the use of commercial starters from
The MLF is not always successful even if it is conducted by inoculated commercial
In summary, the use of wild yeast and LAB can be used to define the typicity of the wines of a region. Some authors stated that the microflora diversity is characteristic of a given area and could be considered its microbiological fingerprint [38, 39]. The inoculation of selected wild yeast and LAB species could help to control the development of the AF and MLF and to improve the complexity and could typify the wine of a region .
5. Fermentation end-products and wild microorganisms
Numerous fermentation end-products contribute to the aroma and flavor characteristics of wines, which determine their quality and final complexity. As it is known, wine is made up of thousands of aromatic compounds, and a large part of them are produced or transformed during the AF and MLF . As mentioned above, these processes are carried out by wild or commercial strains. The use of wild strains allows us to obtain wines with a unique expression with representative characteristics of each variety and area. The aromatic profile of wines is determined by varietal aromas (from grapes), fermentative aromas (produced by yeast and LAB during fermentations) and post-fermentative aromas (associated to the aging period). The fermentative aromas clearly influence the final quality of wines, and the strains used during winemaking are responsible for the presence or absence of some flavors and other non-volatile metabolites . Ethanol, carbon dioxide and glycerol are the main fermentative products. Ethanol is the main volatile product of yeasts metabolism, followed by diols, higher alcohols and esters. The ethanol content influences the wine viscosity and contributes on the aroma fastening. Other important metabolites derived from AF, such as pyruvic acid, participate on the formation of secondary products namely diacetyl, keto acids, succinic acid and butanediol . Succinic acid and glycerol are two of the most important by-products affecting the “body” of the wine. Succinic is the main acid produced by yeasts, and its formation is strain dependent. The tartaric acid experiment slightly changes during fermentation, and the malic acid usually decreases during MLF, although the yeasts metabolism can also modify its concentration during the AF. Regarding acetic acid, this compound may reach more than 90% of the volatile acidity, and it is one of the most important by-products that negatively affect sensory profile of wine. This acid is mainly synthetized by acetic acid bacteria but also may be synthetized by yeasts and LAB.
Other volatile acids, such as propionic and hexanoic acids, are also produced by yeasts and bacteria, as a result of the fatty acid metabolic pathway . Another important, but not always desirable, secondary metabolite of wine fermentation is the acetaldehyde. This compound is the product of the decarboxylation of the pyruvate during the AF. The higher alcohols represent another group of secondary products influencing the sensory profile of wines. The concentrations of higher alcohols are influenced by factors such as the yeast strains, the concentration of amino acids (the precursors of higher alcohols), ethanol concentration, fermentation temperature, pH, composition of grape must, aeration, etc. The higher alcohols are also important precursors for the ester formation; both of them are associated with pleasant aromas, although at high concentrations they can be undesirable.
LAB modulate the flavors of wines by modifying their chemical composition and, therefore, its sensory properties. LAB are responsible to maintain the result of the yeasts metabolism and to increase the complexity and microbial stability of the wine . LAB decrease the wine acidity by the decarboxylation of malic acid to lactic acid, and they also contribute to the aroma by metabolizing other acids such as citric acid. The degradation of citric acid produces acetic acid and diacetyl, both of which have an important and undesirable effect on the wine flavor. Other metabolites affected by LAB metabolism, which have an impact on wine flavor, are alcohols, such as glycerol and mannitol, or carbonyls such as acetaldehyde and diacetyl. Finally, esters can also be modified for LAB species, including ethyl acetate, ethyl hexanoate, ethyl lactate and ethyl octanoate . Depending on the species or even on the strains, LAB may be beneficial or detrimental to wine quality . Meanwhile, acetic acid bacteria, as mentioned above, are only spoilage microorganism because they lead to the formation of such major oxidized aromas (acetaldehyde, acetic acid and ethyl acetate).
During the spontaneous AF, the development of many aromatic compounds occurs, mainly those belonging to the families of alcohols, ethyl esters, fatty acids, acetates and carbonyls. Aliphatic esters and alcohols seem to be more influenced than acids and carbonic compounds. In addition, terpenes and norisoprenoids, well-known primary aromas, can be provided by the wild yeasts during fermentation [43, 44]. The main aromatic descriptors of all of them are the fruity and floral notes, always appreciated in wines. The use of wild yeast to conduct a spontaneous AF may produce higher concentration of alcohols (1- hexanol, phenylethanol), terpenes and other aromatic compounds, such as β-phenyl acetate and γ-nonalactone, compared to wines produced by selected yeasts . The selection of indigenous
The most related aromas of the inoculated MLFs are commonly associated with butter, yogurt, sulfur and toasted notes. Moreover, during spontaneous MLFs, the formation of many aromatic compounds is affected. Some studies have demonstrated the biosynthesis of the aromatic compounds produced during this kind of fermentations and its sensory repercussion. A reduction of herbaceous and vegetable aromas has been highlighted, and the appearance of fruity and floral aromas has been reported . The changes produced in the aromatic composition of Tempranillo wines during spontaneous MLF by using wild LAB have been reported, showing significant increase in esters, lactones, terpenes, norisoprenoids and volatile phenols, such as vanillin and furfural .
6. Selection of wild yeast and lactic acid bacteria
For the selection of wild yeasts and LAB species of a specific wine region, first, it is needed to conduct a biodiversity study, knowing which species are present in grapes. After that, knowing the species at the different stages of a spontaneous fermentation, at the beginning, middle and end of fermentation, is essential. The first stage is to conduct a spontaneous fermentation. Then, isolate different colonies at each stage of the fermentation to obtain a collection of the different microorganisms implied. The second stage is to identify and typify each colony. The recovery and the molecular characterization of a high number of yeasts and LAB strains should be considered to establish a strain collection of oenological interest.
For the yeast species identification, different techniques could be applied. The restriction analysis of ribosomal gens is the simplest technique, reliable and extended [10, 49]. Nevertheless, several available techniques, such as microsatellites (SSRs), Rapid Amplification of Polymorphic DNA (RAPD-PCR), Pulsed-Field Gel Electrophoresis (PFGE) and DNA array technology, have been used to typify yeast strains. Between all these techniques, the more usual techniques for their simplicity and reproducibility are the restriction analysis of mitochondrial DNA  and the amplification of delta elements . PCR-based methods have been already successfully used to identify LAB in different wines. To identify different LAB species, a good technique, fast and reliable, is the Restriction analysis of the amplified 16S-rDNA (ARDRA-PCR) [52, 53]. RAPD-PCR (Random Amplified Polymorphic DNA) is considered to be a suitable method to typify
Once the wild species and strains are identified and typified, the next step is to characterize each isolated strain, which has different genetic profile between them and between commercial yeast. Performing micro-fermentations with pure inoculations of all the strains with the specific characteristics of wine region must in order to test relevant species starter kits. With a better understanding of the different yeasts properties, the yeast selection procedure can be adapted to acquire strains that could improve the wine quality . The AF and MLF performance by selected strains at winery conditions is the last step of selection.
The wild starter kit can be a single strain of
The LAB selection as starters requires an ecological study and the characterization of useful technological and physiological features of the isolated strains in order to select the ones that are potentially more suitable for industrial applications. The selection of LAB for wine inoculation is essentially based on the survival of this strain and the consumption of malic acid. However, there are other important properties that are required to study the ability to produce biogenic amines and different enzymatic activities related to the final aroma profile.
The selection of microorganisms has been successfully used to improve the technological properties of different wines as well as their sensorial profiles helping in the production of wines without sulfites, reducing the levels of ethanol, increasing the glycerol content, varying the acidity of the must and realizing different aromatic components. In summary, the selection of wild yeast and LAB offers the best way to obtain different species and strains, which could improve the oenological characteristics and sensorial profile of wines, giving tools to the oenologist to direct their wine fermentation process. The exploitation of the microbial diversity that exists in the vineyards and in the cellars with the selection of wild yeast and LAB strains has been considered an interesting approach to overcome the distinctive peculiarities of wines produced in different regions .
7. Wild yeasts and lactic acid bacteria from viticultural Spanish regions
This section summarizes several studies carried out in VITEC (Wine Technology Center) from different grape varieties and Spanish regions. The isolation, identification and selection of wild yeasts and LAB were performed in 2016 and 2017 vintages, from grapes and spontaneous fermentations. The grape varieties studied were Verdejo from D.O. Rueda, Albariño from D.O. Rías Baixas and Tempranillo from D.O. Ribera del Duero and D.O.Q. Rioja. In all the cases, different species of
R. del Duero
Ribera del Duero
In order to study the influence of fermentation mixtures from wild selected yeasts on the wine properties, several studies were carried out in VITEC. The behavior of all yeast strains were studied, conducting fermentations at laboratory scale and at semi-industrial scale, in pure (
Concerning LAB, the highest diversity was found at the beginning of the AF. Up to eight different wild LAB species and fourteen
Wild yeasts and lactic acid bacteria are interesting microorganisms that contribute to differentiate the wine character. The suitable use of numerous wild species and strains during winemaking favors the improvement of the complexity and the organoleptic properties of wines. The production of wines by spontaneous or inoculated fermentations using selected wild microorganisms is a remarkable practice for wineries. Further studies should be done in order to deep into the knowledge of the wild microflora of grapes and wines to better understand their behavior and importance. Even more, some drastic physical and chemical winemaking techniques, increasingly used in wineries, could be replaced taking advantage of the biological properties of these microorganisms. Above all, wild yeast and lactic acid bacteria may help to produce modern and new wine styles in a climate change viticultural environment.
We thank MINECO and CDTI for their financial support through the projects MICROSEL (RTC-2016-5559-2) and GLOBALVITI (CIEN Program Ref.IDI-20160746), respectively. Thanks bodegas Matarromera S.L. (Valbuena de Duero, Valladolid, Spain) and Martín Codax SA (Cambados, Pontevedra, Spain) for supplying samples.
Conflict of interest
We have no conflict of interest to declare.