Update on the Role of Saccharomyces cerevisiae in Sherry Wines

1. Introduction

The practice of biological aging of wines is a traditional method observed in various regions worldwide. Examples of such regions include Jura in France, Szamorodni and Aszú in Tokaj-Hegyalja, Hungary, as well as Vernaccia di Oristano in Sardinia, Italy. However, the most renowned biologically aged wines are notably crafted in the Jerez-Xèrés-Sherry and Manzanilla de Sanlúcar de Barrameda Designation of Origin (D.O.) located in southern Spain [1, 2].

Jerez-Xèrés-Sherry wines undoubtedly represent one of the most acclaimed and globally recognized Spanish national products. This recognition is attributed not only to the unique geographical region but also to the winemaking practices associated with their production, preserved for generations and esteemed to the extent of being considered an international oenological reference. Thus, the wines of the Jerez region encompass a wide range of wines with unquestionable personality and authenticity [3].

In October 2022, the Consejo Regulador of the Denominations of Origin, a public law corporation, implemented changes and modifications. The recent agreement specifies that Fino can only be produced in Jerez de la Frontera or El Puerto de Santa Maria. Sherry wines made in Sanlúcar can exclusively be labeled as Manzanilla. Another significant change in the regulations is that sherry wines can no longer be categorized as fortified wines if they reach a minimum alcohol content of 15% (v/v ethanol) without the addition of alcohol. These modifications also involve the utilization of several outdated local pre-phylloxera grape varieties for sherry production, notably the ancient varieties Mantúo Castellano, Mantúo de Pilas, Vejeriego, Perruno, Cañocazo, and Beba. These adjustments and considerations offer winemakers the opportunity to prepare for the future while innovating within the longstanding tradition of sherry production [4].

The production of Sherry and “Manzanilla” wines involves a two-step process. Initially, they must undergo alcoholic fermentation by commercial or naturally occurring yeasts on the surface of Vitis vinifera var. Palomino Fino grapes, resulting in the production of a “young” wine. Young base wines are crafted from grapes cultivated in nine different regions, including Jerez de la Frontera, Sanlúcar de Barrameda, and El Puerto de Santa Maria. Additionally, wines are produced in the wine cellars of Trebujena, Chipiona, Chiclana, Rota, Puerto Real, and Lebrija, which are located in Seville [1, 2, 4].

Subsequently, this base wine undergoes biological aging, facilitated by other specific yeasts known as flor yeasts. Biological aging comprises two distinct phases. The first phase, known as ‘añadas’ or static aging, involves storing the wine in a barrel (known as “bota”) for several years. This static phase is followed by a dynamic phase called “criaderas-solera” or “soleraje,” which consists of a series of oak barrels containing sherry in various stages of maturation (Figure 1).

The Jerez barrels, painted in matte black for easy identification of leaks and with a capacity of 600 liters (equivalent to 36 arrobas), are the most common in the wineries of the Sherry region, although there are also different sizes such as the small butt (500 liters), the half butt (250 liters), or the hogshead (700 liters), among others. In the past, Sherry wineries used different types of wood, including cherry, chestnut, and oak. However, it was oak that turned out to be the best choice for making, storing, and transporting wine. This preference for oak was partly due to the thriving trade with the New World, where oak was readily available. Ships that carried various goods, including wine in barrels (which was essential for trade and as a food source for the crews), often brought back oak wood from the Americas on their return voyages [5]. Today, Jerez wines are primarily aged in American white oak barrels, although some are also aged in barrels made from Spanish or French oak (Figure 2).

The initial stage, referred to as “sobretablas,” occurs when the wine is young and contains approximately 15.5% (v/v) ethanol. The intermediate stages are called “criaderas,” while the final stage, known as “solera,” contains the oldest wine. From the solera, the finished wine is withdrawn. However, the 600 L barrels are only partially emptied during each withdrawal, never exceeding one-third of their contents. The transfer of wine from one oak barrel to the next is termed “rocío.” This process typically occurs twice a year. A common characteristic of these barrels, in the case of Fino and Manzanilla wines, is the presence of the veil of flor, approximately 1–3 cm thick, formed by yeasts and other microorganisms that grow on the free surface of the wine (Figure 3).

2. Yeast diversity in sherry wines during biological aging

The predominant yeast species responsible for the film formation in Sherry wine aging is Saccharomyces cerevisiae. Once the study of flor yeasts began a few 100 years ago, it was observed that these yeasts were different from the rest of the wine yeasts belonging to the same species. Indeed, they have been able to adapt to a really hostile environment, such as the absence of easily fermentable sugars and the presence of high concentrations of ethanol and other toxic compounds such as glycerol, acetaldehyde, or acetoin [6].

These yeast strains are able to carry out an oxidative metabolism, being in contact with oxygen. This biofilm formation required genetic divergence from the S. cerevisiae fermentative strains, with differential expression of genes related to the regulation of sugar metabolism and tolerance to osmotic stress, crucial mechanisms in this particular winemaking context [7, 8, 9].

It is important to highlight the intrinsic genomic diversity of S. cerevisiae flor strains. Recent studies have revealed a remarkable genetic heterogeneity in this subspecies, evidencing a rich pool of genetic variability that contributes to their versatility and adaptability. In fact, it has been demonstrated that these yeasts have a common phylogenetic origin that is distinct from the rest of the wine S. cerevisiae yeast strains [10, 11, 12].

Specific alleles and genetic variants have been identified in genes associated with the synthesis of key enzymes in biological aging, as well as in those related to the response to cellular stress induced by external factors, such as high ethanol concentration and nutrient limitation. For instance, a 24 bp deletion in the ITS1 region has been found in flor strains isolated in Spain and Italy, or a “C” insertion in the same region in these isolated in France [13, 14, 15].

The FLO11 gene in S. cerevisiae flor yeasts plays a key role in the formation and maintenance of the flor veil, a distinctive feature of this group of yeasts. This gene codes for a surface adhesion protein that allows yeasts to bind and subsequently form a matrix that floats on the surface of the wine during biological aging. This matrix acts as a protective barrier that isolates the yeasts from the liquid medium and air, allowing them to survive in adverse conditions while maintaining their proper activity. Flor yeast strains are known for a distinct 111 bp deletion in the promoter region of the FLO11 gene, a crucial adhesion gene responsible for cell aggregation and film formation. This deletion is common among sherry strains from Spain, France, Italy, and Hungary. Its role is to deactivate long noncoding RNAs, specifically ICR1, which represses FLO11 transcription. Disabling this negative regulator, ICR1, stimulates the production of Flo11 protein in flor yeast. The FLO11 coding sequences in S. cerevisiae can vary from 3 to 6 kb, primarily due to differences in the number of repeats in the central domain. Initially, it was believed that Sherry yeast strains encoded longer FLO11 variants with higher hydrophobicity. However, subsequent research revealed that the central domain of FLO11 is notably unstable under non-selective conditions, therefore it seems that one of the factors for the emergence of S. cerevisiae flor yeast may be due to the presence of other repeats, which in turn influence the level of expression of the FLO11 gene [7, 16, 17, 18]. FLO11 sequence is registered in the Yeast Genome Database at Stanford University Medical School (www.yeastgenome.org). FLO11 differs from all other known cell surface flocculins in that it is located near a centromere rather than near a telomere, and its expression is regulated by mating type.

Currently, researchers employ comparative genomic and transcriptomic to unravel the molecular mechanisms behind the transition of an S. cerevisiae strain into a flor yeast. This approach helps identify changes in gene expression associated with unique cell adhesion, stress resistance, iron uptake, nitrogen and carbon metabolism, lipid metabolism, and the production of aromatic compounds. In this sense, other genes that have been found to be directly related to the success of S. cerevisiae flor yeasts include BTN2, HSP12, YAP1, ACC1, FRE2, MCH2, and YKL222C [11, 19, 20]. However, more research is still needed to better understand the modifications of these genes and others that still remain unexplored involved in this process to better understand the mechanism of adaptation and/or domestication of this particular yeast strains to this specific niche. All of these factors are pivotal in the distinct function of veil-forming yeast. These modern techniques are getting insights into the yeast’s adaptation mechanism and enable the development of genetic markers for selecting suitable strains for sherry production [3, 21].

Traditionally, flor yeast strains have been classified based on their ability to metabolize different sugars, categorizing them into four varieties or races within the species Saccharomyces cerevisiae. However, this classification is considered outdated and even simplistic, as significant differences have been found in wines produced in neighboring wine casks from the same base wine [3]. Additionally, it is worth noting that traditionally, in the wine industry, as a whole and particularly in the biological aging of Sherry wines, yeast strains belonging to different species of S. cerevisiae capable of forming biofilm were considered undesirable and even contaminants. However, interest in these yeasts has been growing exponentially due to their potentially valuable phenotypic characteristics.

In a recent study on the diversity of the veil of flor in the Jerez region, four different species (Wickerhamomyces anomalus, Pichia membranaefaciens, Pichia manshurica, and Pichia kudriavzevii) were found in addition to S. cerevisiae flor strains during the biological aging of sherry wines (Table 1). Although they were isolated sporadically from some of the casks analyzed, their origin remains unclear [27, 28]. However, their phenotypic characteristics, such as a good capacity to aerobically assimilate ethanol, glycerol, or urea, together with their ability to form biofilm or the formation of an extracellular matrix, suggesting that they must necessarily be adapted to the specific and difficult environment of sherry wines while maintaining an active growth rate. A study carried out in the Montilla-Moriles region (Cordoba, Spain) has also confirmed the presence of some of these non-Saccharomyces species, as well as others not previously described in this system, such as Candida guillermondii and Trichosporon ashaii, using regrowth technique to increase their concentration prior to their identification, which was carried out using Next Generation Sequencing (NGS) techniques [12]. The same research group has recently described the presence of 26 other previously undetected microorganisms in the system, hinting at the possibility that their presence may be due to the mites and flies present in the cellars. Regardless of their origin, and despite the growing number of studies focusing on the identification of various non-Saccharomyces yeast species and strains, the potential of many of these species remains largely undiscovered, and they could be of great interest not only in the context of biological aging, but also from a wider biotechnological point of view [12].

3. Other microorganisms distinct from yeasts in the veil of flor

During the biological aging process of Sherry wines, various other microorganisms can coexist with the yeast strains responsible for veil formation. These include lactic acid bacteria from the genera Leuconostoc, Pediococcus, and Lactobacillus, as well as opportunistic fungi like Botrytis cinerea, Penicillium chrysogenum, and fungi of the genus Rhizopus. The presence of lactic acid bacteria, such as Lactobacillus hilgardii, Lactobacillus plantarum, and Lactobacillus brevis in Sherry veils of Flor, is closely associated with the high levels of gluconic acid produced by the filamentous fungi B. cinerea [1, 29, 30].

The species S. cerevisiae and related yeasts are susceptible to various intracellular nucleic acid infectious agents, including dsRNA viruses, ssRNA viruses, LTR retrotransposons, and bicatenary DNA plasmids. These infectious agents share certain similarities with higher eukaryotic viruses. For example, yeast retrotransposons exhibit a life cycle akin to retroviruses, the structure of yeast Totiviruses mirrors the capsid of reoviruses, and yeast plasmid segregation resembles viral episome segregation strategies (Figure 4).

Modern experimental tools available for studying the genetics, cell biology, and evolution of S. cerevisiae and other yeasts are well-suited for expanding our knowledge of how these intracellular agents exploit cellular processes. Importantly, it remains unclear whether yeast-related issues in alcoholic fermentation or premature weakening of the flor veil in wines could potentially be triggered by viral infections, representing a significant area for further investigation. A recent study conducted in our laboratory revealed that flor yeasts infected by Mycoviruses develop a killer factor that facilitates their dominance in the formation of the biofilm (data not shown). This could provide an evolutionary advantage, particularly in environments with high ethanol concentrations.

The presence of this microbiota, distinct from yeast, can lead to the development of undesirable wines and an accumulation of high concentrations of biogenic amines. However, the proliferation of some yeast species like S. cerevisiae, can also potentially introduce sensory deviations and negatively impact the wine’s quality.

4. Off-flavors and carcinogenic agents in sherry base wines; solutions to eliminate them from a microbiological and biotechnological perspective

Current studies showed that the base wine plays a more substantial role in Sherry wine production than previously believed. This suggests the potential for substantial enhancements in yeast strains that have already been chosen and are used in industrial processes. These improvements can be achieved without the need for specific genetic modifications; instead, they leverage the genetic diversity present within the yeast strain population. This way, the base wine (i.e., young white wine) used for the production of various Sherry wines can undergo not only sensory improvements but also enhance food safety, which will substantially impact the resulting young white wine and, consequently, the final product [21, 31].

Hydrogen sulfide is a metabolite associated with an aromatic defect in wines (rotten eggs) and has a very low detection threshold. Consequently, its reduction has been extensively studied in yeast but is not yet employed on an industrial scale. On the other hand, urea spontaneously reacts with ethanol to form ethyl carbamate, a genotoxic substance. The generation of this metabolite is highly favored by rising temperatures and high ethanol concentrations, so its presence in biologically aged wines is expected to increase as global warming raises temperatures [21].

In our laboratory, we conducted a study with the aim of reducing the production of hydrogen sulfide (H2S), a compound associated with an undesirable aroma defect. Two distinct improvement strategies were employed. Firstly, a mass mating technique was used, involving the hybridization of the target strain with another parental strain known for its low H2S production. Hybrid strains were selected based on complementary auxotrophic markers (inability to grow on melezitose and galactose, respectively) exhibited by both parental strains. Additionally, a distinct transposon amplification profile allowed the identification of putative hybrids with profiles intermediate between the two parental strains [21].

Furthermore, adaptive laboratory evolution was employed using ammonium molybdate as a selective pressure, an analog toxic to sulfate. Before applying the selective pressure, the yeast strain was sporulated, ascospores were dissected, and mating was allowed to increase the genetic variability within the population.

In both cases, rapid and qualitative screening of variants was made possible through the use of specific culture media (BiGGY). In total, 10 yeast variants were obtained (2 hybrids and 8 variants through adaptive evolution) that met basic oenological parameters and exhibited phenotypic characteristics similar to the parental yeast. Following pilot-scale fermentations in both synthetic and natural grape musts, three candidate variants were selected for industrial-level fermentations: two hybrids and one variant obtained through adaptive evolution.

Once produced at an industrial scale, the wines underwent analytical and sensory evaluation. Wines produced using the variants obtained through hybridization exhibited greater differences from the control strain in terms of terpenes and ester production, as expected, while the evolved strain closely resembled the parental strain. As a result, not only were wine defects reduced at an industrial level, but the wines were also noted for their enhanced floral and fruity characteristics.

On another note, efforts were made to reduce urea excretion during alcoholic fermentation, as urea is the main precursor of ethyl carbamate. For this, only the adaptive evolution technique was employed in the laboratory, similar to the first study, using L-canavanine as a selective pressure, a toxic analog of l-arginine [31].

Following screening in the laboratory, a variant was selected for industrial wine production, resulting in reduced urea production and improved fermentative capacity of the yeast strain. The wines also exhibited significant differences in both chemical composition and sensory analysis, once again being favorably rated by tasters compared to those produced with the parental strain [31].

5. Mitigating the consequences of climate change on the veil of flor formation

On the other hand, biological aging is a highly complex and delicate process. The stability of the yeast of flor biofilm depends on various factors, with the environmental conditions of the specific winery being among the most defining.

Studying yeast populations that make up the flor film using molecular techniques and analyzing their behavior during aging can provide valuable insights to better understand the process. Research is crucial for developing strategies to maintain the identity of these wines despite viticulture changes due to the effects of climate change. Additionally, it is assumed that the yeasts present in this biological aging system, whether or not they belong to the S. cerevisiae species, can be very interesting from a biotechnological perspective, as they have had to adapt to very adverse conditions to remain metabolically active.

The effects associated with climate change, such as rising temperatures, increased carbon dioxide levels, and a significant reduction in rainfall, are directly impacting the wine industry as a whole, with a particular focus on the Jerez region. As a consequence of rising temperatures, excessive grape ripening occurs, leading to two direct effects on the must’s composition: increased sugar concentration and reduced easily assimilable nitrogen content. This imbalance can cause deviations during the base wine fermentation, even when using commercial or selected yeast strains, resulting in an increase in undesirable compounds in the wine, such as hydrogen sulfide or urea, as stated before.

Furthermore, it is known that the stability of the veil of flor largely depends on the wine’s ethanol content and cellar climatic conditions, including temperature and relative humidity. Therefore, the effects of climate change directly impact the biological aging process, progressively weakening the biofilm’s structure, composition, and thus its activity. This can lead to the disappearance of the flor film from the wine’s surface, causing not only oxidation but also a noticeable sensory deviation in biologically aged wines.

Despite the existence of classic methods for regenerating the flor veil through direct grafts from a barrel with a healthy biofilm, it has been observed that the time it takes for this veil to re-form is often lengthy. As a consequence, there is some oxidation of wines undergoing biological aging, leading to a loss in their quality. Therefore, we aimed to apply new techniques that would contribute to the development and implementation of targeted flor veils.

We optimized a culture medium based on ethanol as the primary carbon source, allowing for the fastest possible biofilm formation. Once we obtained the culture medium that enabled faster flor veil formation, we developed a support printed with a 3D printer using polylactic acid (PLA), a biodegradable material relatively stable in acidic environments and with high ethanol concentrations. This latter prototype has resulted in the publication of a patent (ref. P202130692).

6. Searching for an alternative to wine ethanol in wine fortification

One of the key characteristics of Sherry wines is their classification within the category of fortified or “encabezados” wines. For their production, it is essential to use alcohol to increase their alcohol content above 15% vol. The relevant European legislation (Regulation [EU] 2019/934 of March 12, 2019) and the Specification of the Designation of Origin (D.O.) restrict the type of alcohol allowed for this category of products, exclusively authorizing alcohol obtained through the distillation of wines.

For the fortification, topping, or enrichment of wines protected by the D.O. Jerez-Xérès-Sherry, wine alcohol with an alcohol content ranging between 95.0 and 96.0% (v/v) is used, as stipulated by the relevant legislation. However, the impact of using alcohols derived from raw materials other than wine alcohol on the microbial populations present in wines aged under biological aging conditions (Finos) is still unknown, as well as its effect on the generation of volatile components and its influence on the sensory profile of these wines. Therefore, we proposed an alternative approach for fortifying wines using alcohols obtained from malt, grape pomace, agave, sugarcane, and cereal, as well as wine alcohols obtained through various distillation techniques. The results may reveal alternatives to the current use of grape-derived alcohol, potentially impacting both product quality and cost-effectiveness (results not shown).

Ongoing experiments at winery scale regarding the use of non-grape-based alcohols at different alcohol levels than currently authorized for fortifying base wines represent a variable that could potentially optimize the biological aging process of Fino wines. Initial results reveal significant differences in the speed of development and morphology of S. cerevisiae and P. kudriavzevii yeast biofilms for certain tests, as well as variations in the volatile composition of the aging wines [32].

It is also expected that the sensory and compositional contributions of the different alcohols will impact the organoleptic profile of Fino wines. Additionally, these alcohols provide alternative carbon sources to the flor yeast, which could be of great importance if the yeast assimilates these nutrients through metabolic pathways different from the usual ones. This could result in the formation of compounds that add distinctiveness and quality nuances to the wine produced through biological aging.

7. Conclusions

The production of biological-aged Sherry wines is a distinctive process that relies on the veil of Flor. The maintenance and storage of these cultures play a crucial role in this process, leading to the development of unique low-molecular compounds responsible for the distinct flavor and aroma of sherry wines.

Recently, numerous countries worldwide have begun adopting microbiological and food technology to produce Sherry-like wines. In our previous research, we endeavored to create a screening method for identifying promising veil-forming yeast strains to craft Sherry-like wines.

This chapter has shown that the veil of flor requires careful consideration of essential winemaking factors and contributes to our comprehension of the evolution of flor yeast. Furthermore, it aids in enhancing sherry production technology by integrating new microbial compositions into industrial strains.

On the other hand, biotechnological methods for yeast improvement focus on enhancing genetic diversity naturally through sexual reproduction. Directed evolution, sometimes combined with mutagenic agents or sexual hybridization, increases genetic and phenotypic variation for selection. Laboratory S. cerevisiae strains are usually preferred due to their haploid nature and better understanding. However, applying these techniques to industrial strains is challenging due to variable characteristics like sporulation efficiency and spore viability, common in the laboratory but not in industrial settings. Industrial strains are often polyploidy, lack suitable markers, etc.

In the context of climatic change and biological aging as well, the PLA-printed prototype has proven to be a powerful scaffold for veil of flor yeast at the laboratory scale. This allows for grafting to occur more quickly than free yeast cells can achieve. Nevertheless, further investigation is required to assess the stability of these scaffolds in acidic environments and under increased temperatures, among other potential factors they may encounter.

Flor yeast strains remain relatively understudied even today. Therefore, the aim of this chapter has been to provide an updated overview of these yeast strains as of the year 2023. We hope that this chapter will encourage further research and investigations into these fascinating microorganisms, particularly within the context of Saccharomyces cerevisiae.

Acknowledgments

We greatly appreciate the cooperation of Bodegas Fundador in providing samples for this study. We also thank Bodegas Ligures (Mesas de Asta) and Francisco Núñez for giving us access to the mural of the parts of the barrel, Jesús Pérez Chicón for his rewarding help with Mycoviruses experiments, and Manuel Fernández Barcell and Jesús Cantoral for their photos.

Conflict of interest

“The authors declare no conflict of interest.”