The Life of Non-Saccharomyces Yeasts in Wine

1. Introduction

The transformation of fruit juice into wine through alcoholic fermentation is indeed a fascinating process that has been studied by scientists for many years. This transformation occurs due to the action of yeasts, which convert the sugars present in the fruit juice into alcohol and carbon dioxide [1]. Yeasts are unicellular microorganisms that are found in abundance in nature and play an essential role in the fermentation of many foods and beverages, including bread, beer, and wine [2]. During the fermentation process, yeasts consume the sugars present in the fruit juice and produce alcohol as a byproduct. The most common yeast species used in wine fermentation is Saccharomyces cerevisiae, which is capable of fermenting both glucose and fructose, the two main sugars present in grapes [2]. The choice of the yeast strain used during fermentation can have a significant impact on the final wine product’s aroma, flavor, and overall quality [1,3]. In addition to yeasts, lactic acid bacteria (LAB) also play a secondary role in the wine fermentation process [4,5]. LAB are responsible for the malolactic fermentation, a process that occurs after alcoholic fermentation and results in the conversion of malic acid into lactic acid. This process can impact the acidity and overall taste of the wine [6]. Thanks to the pioneering work of microbiologists such as Antonie van Leeuwenhoek and Louis Pasteur, the scientific community has a better understanding of the wine fermentation process [7]. With the advancements in scientific knowledge, winemakers and oenologists can now use this knowledge to produce high-quality wines by selecting appropriate yeast strains, controlling the fermentation temperature, and monitoring the fermentation process [2]. Throughout the winemaking process, critical decisions need to be made that impact the fermentation process. Perhaps the first and most challenging decision is whether to add selected yeasts or to rely on the indigenous microbiota to drive the process. While most experts favor adding selected yeasts, allowing the indigenous yeasts to modulate the final wine creates its unique characteristics, setting it apart from other wines [8,9,10,11,12]. Winemakers must be aware of the yeasts present at each stage of production, including at harvest and during the start, mid, and end stages of fermentation [13,14,15,16,17,18]. It is now universally recognized that to produce quality wine, the joint participation of S. cerevisiae and various other yeasts is required, either by their natural participation in vivo or through the addition of commercial preparations containing their lysates [3,19] (Figure 1).

In this review, we will discuss the various yeasts involved in wine fermentation and how they interact to produce a high-quality end product that is appreciated by consumers.

2. Saccharomyces cerevisiae

In addition to Saccharomyces cerevisiae, there are many other yeast species that can be found in grape must and can have an impact on wine quality. Non-Saccharomyces yeasts, such as Hanseniaspora and Metschnikowia, are known to produce a range of aroma and flavour compounds, including esters and higher alcohols, that can contribute to the complexity of wine [20,21,22,23]. However, they are generally less tolerant to high levels of alcohol and can be outcompeted by S. cerevisiae during the fermentation process, leading to incomplete or stuck fermentations.

Recent research has focused on the potential of using mixed cultures of Saccharomyces and non-Saccharomyces yeasts to improve wine quality. By combining the desirable characteristics of different yeast species, it may be possible to produce wines with more complex and unique flavour profiles. However, the use of mixed cultures can also introduce additional complexity to the fermentation process, as the interactions between different yeast species can be difficult to predict [24,25].

Overall, the choice of yeast strains used in winemaking can have a significant impact on the chemical and sensory properties of the final product. Winemakers must carefully consider the desired flavour profile and fermentation characteristics when selecting yeast strains, and may choose to use mixed cultures to achieve specific goals. Ongoing research in this area may lead to new insights and techniques for optimizing wine quality through yeast selection and fermentation management [26,27,28,29,30].

3. Non-Saccharomyces yeasts

Furthermore, recent research has shown that non-Saccharomyces yeasts can also con-tribute to the sensory characteristics of wine. For example, some non-Saccharomyces yeasts can produce volatile compounds that contribute to fruity, floral or spicy aromas in wine [31,32,33]. Others can produce enzymes that release aroma precursors, which can lead to the development of complex and desirable aromas during aging. Non-Saccharomyces yeasts can also contribute to the mouthfeel of wine by producing polysaccharides and glycerol, which can increase the viscosity and perceived body of the wine [34].

In addition to wine production, non-Saccharomyces yeasts are used in the production of other fermented foods and beverages, such as beer, cider, mead, and kefir. In beer production, non-Saccharomyces yeasts can contribute to the flavor, aroma, and mouthfeel of the final product.[35,36,37]. They can also improve fermentation efficiency and reduce the risk of contamination by spoilage microorganisms. Similarly, in cider and mead production, non-Saccharomyces yeasts can contribute to flavor and aroma complexity and improve fermentation efficiency. In kefir production, non-Saccharomyces yeasts can contribute to the texture and flavor of the final product [24,38].

Overall, the use of non-Saccharomyces yeasts in fermentation processes can have a significant impact on the sensory, chemical, and microbial properties of the final product (Table 1). With the help of molecular techniques, researchers can better understand the diversity and functionality of non-Saccharomyces yeasts and their potential applications in various fermentation processes. It is worth noting that the use of non-Saccharomyces yeasts in winemaking requires careful consideration and control. The use of these yeasts can result in the production of off-flavors, such as hydrogen sulfide, which can affect wine quality. Additionally, these yeasts have varying fermentation kinetics, which can impact the duration and success of the fermentation process. As such, winemakers must carefully select the appropriate non-Saccharomyces yeast strains for their specific winemaking goals and monitor the fermentation process closely to ensure optimal results [39,40,41].

Overall, the use of non-Saccharomyces yeasts in winemaking is a promising area of research that has the potential to enhance wine quality and reduce the use of synthetic additives. However, further studies are needed to fully understand the mechanisms behind the interactions between different yeast species and their impact on wine quality. In addition to their potential to improve the sensory quality of fermented beverages, non-Saccharomyces yeasts have also been shown to have potential health benefits. For example, some non-Saccharomyces yeasts have been found to produce high levels of β-glucans, which have immunomodulatory properties and have been shown to improve gut health [83]. Other non-Saccharomyces yeasts have been reported to produce anti-inflammatory and anti-cancer compounds, such as exopolysaccharides and peptides [84,85]. Therefore, there is increasing interest in the use of non-Saccharomyces yeasts in the development of functional foods and nutraceuticals. Furthermore, the use of non-Saccharomyces yeasts in fermentation has environmental benefits, as they can reduce the use of chemical additives and decrease the carbon footprint of the fermentation process [39]. In addition, some non-Saccharomyces yeasts have been shown to have a positive impact on soil health, as they are able to solubilize soil nutrients and enhance plant growth [86].

Overall, the use of non-Saccharomyces yeasts in fermentation has significant potential for improving the sensory quality, health benefits, and environmental sustainability of fermented beverages and other products. Further research is needed to fully understand their fermentative capabilities and the mechanisms underlying their health-promoting properties [60,87,88].

3.1. Schizosaccharomyces pombe

Initially considered a spoilage yeast, Schizosaccharomyces has been successfully used industrially in the fermentation of cane sugar during rum making, palm and cocoa fermentation, and there are high hopes for its use in the wine industry [42]. Malolactic fermentation is often used to reduce the malic acid content in musts and wines, especially in the production of red wines, although it is sometimes a very complicated process due to the growth requirements of the bacteria used [42]. The use of Sc. pombe could become an invaluable new tool for grapes from wine-growing regions where malic acid is present in excessive concentrations, although few commercial strains are available due to their high rate of acetic acid production (about 1 g/L). Mixed and sequential cultures with Saccharomyces have been used to reduce the negative effects of Schizosaccharomyces spp. strains [89]. However, Sc. pombe has much greater potential than just its ability to reduce malic acid content and ferment sugar. Some researchers are using Sc. pombe to decrease gluconic acid [43]. Another application is aging on lees due to the greater autolytic release of polysaccharides than with Saccharomyces [44]. The ability of Sc. pombe to reduce 4-ethylphenol in wine due to its high adsorption capacity has also been studied [45]. Furthermore, urease activity is also of great interest in relation to food safety. Urea is the main precursor of ethyl carbamate, so reducing urea content could reduce ethyl carbamate, which is one of the main food safety problems in modern oenology[46]. Furthermore, the use of Schizosaccharomyces could limit the risk of biogenic amines [89], which are notorious for causing physiological problems in humans [90]. In addition, Schizosaccharomyces produces a large amount of pyruvic acid, and the significant hydroxycinnamate decarboxylase activity of Schizosaccharomyces favors the formation of vinylphenolic pyranoanthocyanins [89,91].

3.2. Pichia kluyveri

The various species of Pichia are fascinating non-Saccharomyces yeasts in oenology and are typically present in must fermentations, directly linked to wine [57]. The most frequently cited species in literature are P. fermentans [76,77], P. membranifaciens [92], P. occidentalis [93], P. terricola, P. manshurica, P. kudriavzevii, and P. kluyveri. The frequency of Pichia in grapes is lower than that of S. cerevisiae (28%) and other species such as Hanseniaspora uvarum (44%). The frequency varies from 0.12% for P. occidentalis to 4.7% for P. anomala. Other Pichia species commonly isolated from grapes are P. manshurica (2.81%), P. membranifaciens (0.98%), and P. kudriavzevii (0.85%) [94]. As a result, there are no adequate selection methods available, mainly because of these low frequencies that do not contribute to the development of commercial strains. However, P. kluyveri is perhaps the most researched species, for which commercial preparations are available. These species are known for their ability to enhance the composition of aromatic compounds, providing thiols, terpenes, and fruity esters. When added to the fermentation process, they enhance rose petal and floral aromas, contributing to the overall bouquet of the wine, and the varietal and thiol aromas [74,75]. Marketers of wine suggest a sequential use of starter cultures, adding P. kluyveri first and after 48 hours of fermentation, S. cerevisiae, which is better adapted to high ethanol concentrations and will complete the fermentation process. Biochemically, Pichia species ferment glucose but not other sugar molecules easily. Overall, the various Pichia species are gaining increasing interest in oenology [87,95,96].

3.3. Torulaspora delbrueckii

Torulaspora delbrueckii is one of the most commonly used non-Saccharomyces yeasts in oenology. It is recognized for its ability to improve the quality of wine and significantly reduce volatile acidity, particularly in musts with high sugar concentrations [97]. Wine-makers consider this yeast to be a good option for optimizing certain wine parameters, as compared to those produced with S. cerevisiae. Specifically, T. delbrueckii is attributed with a lower acetic acid production capacity, lower ethanol concentration, higher amounts of glycerol, increased release of mannoproteins and polysaccharides, and greater potential for malolactic fermentation. In addition, this yeast has been observed to produce a higher number of desirable aroma compounds such as fruity esters lactones, thiols, and terpenes, while reducing the production of undesirable aroma compounds such as higher alcohols [33,98,99]. From an organoleptic perspective, T. delbrueckii contributes significantly to the reduction of esters and, at the same time, increases the concentration of minor esters and lactones, making it an important factor in the production of white wine [97]. In this way, the action of T. delbrueckii reduces the intensity of the fresh fruit aromas that are characteristic of young wines. At the same time, it increases the aromas of raisined fruit and sweetness, giving the wine aromatic characteristics associated with wines that have been produced over a longer period of time [20]. For this reason, some wine producers consider that T. delbrueckii is not a recommended yeast for making young white and rosé wines, as they are less aromatic and more evolved than those produced with Saccharomyces. The effect attributable to T. delbrueckii varies depending on its degree of involvement in fermentation. When S. cerevisiae is involved, it is usually weak. To increase its effect, killer strains of T. delbrueckii are used, thus achieving a more reproducible effect of these yeasts on the final aroma of the wine [68]. It is known that T. delbrueckii has a lower fermentation potential and a lower growth rate than S. cerevisiae when used under normal fermentation conditions. When using killer strains, the environmental conditions are modified. On the other hand, red wine production differs in certain aspects that may affect the growth of T. delbrueckii during fermentation and the quality of the wine. Frequent punching (oxygenation of the grape skins) allows for a higher availability of oxygen in the fermentation of red wines in comparison with white wines. This may benefit T. delbrueckii, since respiration is more relevant to its metabolism than that of S. cerevisiae, so the former grows worse than the latter under strictly anaerobic conditions [100]. Whereas, the alcohol content is usually higher in red wines than in white wines. This negatively affects the ability of T. delbrueckii to dominate and complete fermentation, especially in the presence of S. cerevisiae, which is a yeast better adapted to high ethanol levels. Nevertheless, the initial amount of wild microorganisms is usually higher in red wine fermentation than in white wine fermentation. Red wine fermentation takes place in the presence of skins, which hold together more bacteria that carry out malolactic fermentation [5]. In addition, the presence of skins in the must provides additional nutrients, which can improve the fermentative capacity of T. delbrueckii and make it more competitive with S. cerevisiae.

3.4. Wickerhamomyces anomalus

The presence of W. anomalus during the fermentation process can have both positive and negative effects on the final wine product. On one hand, research has shown that this yeast contributes to the production of desirable aromas and flavors in wine, such as floral and fruity notes [54]. However, uncontrolled growth of W. anomalus can lead to spoilage and off-flavors in the wine [33,101,102]. To prevent the growth of spoilage yeasts like W. anomalus, sulfites (e.g. sulfur dioxide) are commonly added to the wine by winemakers [103]. The addition of sulfites helps inhibit the growth of unwanted microorganisms, preserving the wine’s freshness and flavor. Nevertheless, some individuals are sensitive to sulfites, and high levels of sulfites in wine can trigger allergic reactions [104]. As a result, winemakers are exploring alternative approaches for preventing spoilage, such as using non-sulfite antimicrobial agents or implementing more frequent monitoring and testing during wine production [105].

Enzymes produced by W. anomalus, such as glycosidases, can contribute to the release of aromatic compounds in wine by hydrolyzing glycosidic bonds that are present in grape precursors [57]. These glycosidic bonds can mask the aromatic compounds, making them less volatile and therefore less perceptible to the human nose. By releasing these compounds, W. anomalus can have a significant impact on the aroma of wine. For example, β-D-glucosidase can hydrolyze glycosides of monoterpene alcohols, which are important contributors to floral and fruity aromas in wine [106]. Strains identified as W. anomalus or its former names have been reported to produce glycosidases such as β-D-glucosidase, α-L-arabinofuranosidase, α-L-rhamnosidase, and β-D-xylosidase, which are involved in the release of compounds aromatics from grape precursors [34].The production of these enzymes by W. anomalus can therefore enhance the aroma profile of wine, potentially making it more complex and interesting. As such, strains of W. anomalus have been studied for their potential use in the oenological industry as a source of enzymes for improving wine aroma.

3.5. Metschnikowia pulcherrima

Metschnikowia pulcherrima is a non-Saccharomyces yeast present in various ecological niches, including the surface of grapes. Morphologically, its shape is ovoid to ellipsoidal with a size of 2.5µm x 4-10µm. The diploid cells of this species propagate vegetatively by budding. Under certain anaerobic conditions it can form pseudohyphae. It can form one to two lance-shaped (acicular/threadlike) spores. Its colonies are cream-colored and produce a reddish-brown soluble pigment called pulcherrimina, characteristic of this species, which gives color to the colonies and diffuses towards the medium. Strains of M. pulcherrima can be identified using selective and differential media: they show positive activity of the enzyme β-glucosidase, expression indicated by the use of arbutin as carbon source in agar plates; and proteolytic activity [107].

It grows well in media such as YPD (yeast extract peptone dextrose) or L-lysine. On the other hand, it shows very weak growth on nitrate agar [58]. Regarding the metabolism of M. pulcherrima, it is known that it can use glucose, sucrose, fructose, galactose and maltose as carbon sources. However, it seems that with lactose it shows weak or non-existent growth. On the other hand, it can grow adequately at low temperatures of 15 to 20◦C and with a pH between 3 and 6 [58]. M. pulcherrima is one of the non-Saccharomyces yeast species capable of expressing more extracellular hydrolytic enzymes, highlighting the following: amylase, cellulase, glucanase, β-glucosidase, β-lyase, lipase, lichenase, pectinase, protease, sulfite reductase, and xylanase [60]. M. pulcherrima is considered a promising candidate for producing wine with low ethanol content. Previous studies have demonstrated that inoculating this yeast in combination with other yeasts, such as Saccharomyces uvarum, can result in wines with lower alcohol concentrations. However, M. pulcherrima has a lower fermentation power compared to other non-Saccharomyces yeasts [58]. Moreover, the reduction in alcohol production appears to be additive when used in combination with other yeasts, but competition and interactions with the autochthonous microbiota of grapes during fermentation can limit the expected results [62].

3.6. Hanseniaspora/Kloeckera spp.

Yeasts belonging to the genus Hanseniaspora are ascomycetes that are easily identified by their characteristic apiculate shape under the microscope, resulting from bipolar budding. This genus is part of the non-Saccharomyces yeasts, which are frequently isolated during the initial stages of fermentation. These yeasts can also be found on the surface of grapes, in soil, and in the winery environment, including harvesting machinery and fruit processing equipment [78,79]. Apiculate yeasts belonging to the genus Hanseniaspora are prevalent on grape surfaces. While H. uvarum is known for its abundant presence on grapes and its negative impact on wine quality due to high volatile acidity production, less is known about H. vineae, which is better adapted to fermentation. Studies have reported that H. vineae has enzymatic activity [79,80] and is capable of producing high levels of desirable aromatic compounds [81,108], enhancing the sensory properties of wines produced on an industrial scale. Additionally, proteolytic activity has been observed in Hanseniaspora isolates [64].

3.7. Lachancea thermotolerans

Lachancea thermotolerans, which was previously known as Kluyveromyces thermotolerans, is a yeast commonly found in various natural environments, including grapes. Its elliptical shape makes it indistinguishable from S. cerevisiae under light microscopy [109]. This yeast reproduces sexually with the formation of 1-4 ascospores and has been available as an active dry yeast since 2012, marketed by CHR-Hansen to enhance the sensory characteristics of wines. A recent review [66] highlights its effects on acidity, aromatic profile, and polyalcohol production. L. thermotolerans has a moderate fermentation power (4-10% v/v) and is recommended for mixed or sequential use with other species such as S. cerevisiae or Sc. pombe, to allow for complete fermentation of the must’s sugars. One of the most noteworthy properties of this yeast is its ability to produce lactic acid, which can effectively enhance the acidity and pH of wines in a stable manner, as this acid remains unchanged during wine aging and stabilization. Additionally, the pH modification capability is crucial, with some strains able to exceed 0.5 pH units under real vinification conditions, particularly when used in sequential fermentations on crushed red grapes, in the presence of solid parts [66]. This is due to the fact that most of the acidification by L. thermotolerans occurs during the initial stages of fermentation, making it a strong competitor against other wine yeast species, and also because of its excellent tolerance to ethanol. In addition to its enzymatic activities, L. thermotolerans also produces a range of volatile compounds that contribute to the aroma of wines. These include esters, such as isoamyl acetate and ethyl lactate, as well as higher alcohols, such as isoamyl alcohol and 2-phenylethanol [67]. It has also been reported to produce sulfur compounds, such as thiol precursors, which can contribute to the tropical and citrus fruit notes in wine [66]. Overall, L. thermotolerans is considered a promising non-Saccharomyces yeast for improving the sensory properties of wines, especially in terms of acidity and aroma. Recent studies describe that it can favor the release of terpenes and volatile thiols [110]. Various works also show a positive effect of the use of L. thermotolerans on glycerol contents [68]. Glycerol is the second quantitatively most important fermentative metabolite after ethanol and has a certain effect on wine smoothness and structure.

Further studies have aimed to explore the biocompatibilities between L. thermotolerans and Hanseniaspora spp. in co-inoculation, using different types of nutrients and considering the effect on Yeast Assimilable Nitrogen, at low temperature (16 ◦C) and medium SO₂ (50 mg/L), for improving the sensory profile [111]. The behavior of these yeasts was evaluated, and significant results were obtained on the population count, with higher populations of Hanseniaspora spp. with respect to L. thermotolerans. Not surprisingly, fermentations with L. thermotolerans/H. vineae, showed inhibition of acidification, generating up to 0.41 g/L of lactic acid. On the contrary, a synergistic effect was observed when L.thermotolerans/H. opuntiae was used, achieving 2.44 g/L of lactic acid and a pH reduction of up to 0.16 [111].

3.8. Candida stellata

Non-Saccharomyces yeasts, such as Candida spp., are becoming increasingly important in the industry due to their unique fermentative behavior. Candida species have been identified as potential candidates for the fermentation of wine and beer [112]. In particular, C. stellata is frequently isolated from grape must and can survive throughout spontaneous wine fermentation for extended periods of time [113]. Studies on the fermentative activity of C. stellata have shown that it can have a positive impact on the taste and flavor of alcoholic beverages [69]. In addition to its ability to positively affect the taste and flavor of alcoholic beverages, C. stellata also exhibits unique metabolic characteristics. It is known to have a strong preference for fructose and high osmotic pressure environments. Under anaerobic conditions or limited oxygen supply, it undergoes alcoholic fermentation, but under completely aerobic conditions, a mixed respiro-fermentative metabolism is observed. This metabolic behavior is influenced by the concentration of oxygen and glucose in the fermentation medium. When conditions are conducive to sugar fermentation, major fermentative compounds such as ethanol, acetic acid, and glycerol are produced, along with small amounts of higher alcohols, esters, volatile fatty acids and carbonyl compounds.

Despite these metabolic losses, the complete fermentation of hexose by yeast can still produce 94–96% of the theoretical yield of ethanol [71,72,73,114].

4. Conclusions

Life in wine is not easy for microorganisms, and the early stages of wine fermentation can be especially challenging. In the first stage, a very sweet juice with high concentrations of sugars provides a placid environment in which yeasts can develop and carry out different metabolic activities, mainly alcoholic fermentation. In such an environment, S. cerevisiae could be the dominant yeast, in the absence of high competition. However, this is often not the case. There are numerous yeast genera present, mainly from other genus grouped under the term non-Saccharomyces, which can outcompete S. cerevisiae in the early stages of fermentation. These non-Saccharomyces yeasts develop and produce numerous aromatic compounds that ultimately define the resulting wine. They are responsible for much of the complex aroma and flavor profiles that we associate with fine wines. This process goes hand in hand with an increasing production of alcohol. However, as the alcohol concentration in the wine increases, the environment becomes more hostile to most yeast species. High amounts of alcohol are unbearable for almost all yeasts, except for S. cerevisiae, which has developed a remarkable ability to tolerate high levels of alcohol. As a result, S. cerevisiae ends up being the triumphant yeast in this world. It is able to outcompete other yeasts and continue to ferment the wine until most of the sugar has been converted to alcohol. During this stage, the yeasts produce more alcohol, and the wine becomes more acidic. Eventually, the alcohol concentration reaches a point where it becomes toxic even for S. cerevisiae, and the fermentation comes to a natural end. The resulting wine is a complex mixture of different aromatic compounds, acids, and other components, all of which contribute to its unique sensory profile.