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While Saccharomyces cerevisiae is recognized as the yeast species that completes the process of alcoholic fermentation during winemaking, the use of starter cultures from other species has become popular in recent years. Non-saccharomyces yeast cultures are now widely used for their bio-protective effects and/or the contribution they make to a wine’s sensory profile. Conversely, starters of wine lactic acid bacteria are also commonly utilized around the same time as commercial Saccharomyces cerevisiae, as an alternative to encouraging adventitious strains to proliferate. This could be either for initiating malolactic fermentation during alcoholic fermentation, or more recently for biological protection of musts prior to the fermentation process. The interactions between S. cerevisiae and other species are documented in the following chapter. The areas examined in more details include requirements of nutrients compared to S. cerevisiae, whether complimentary of symbiotic. Active bioprotective agents such as killer factors, the role of cell-to-cell contact, and the resultant effects on final wine composition when co-fermenting with S. cerevisiae is also discussed.
*Address all correspondence to: gbdha@chr-hansen.com
1. Introduction
While Saccharomyces cerevisiae is generally regarded as the microbial species that completes alcoholic fermentation in wine, it is far from the only species both present and thereby in some way contributing to the process. Winegrapes, the key starting material in winemaking, can contain a diverse range of microbial flora [1], so offer a natural origin for these organisms in a given fermentation. They can also arise from the winemaking environment itself [2] and will continue to be present and viable some way into the winemaking process [3, 4]. Whether colonized on the initial winegrapes or derived from the winemaking environment, typically the populations of species other than S. cerevisiae are naturally far more abundant in the early stages of winemaking than S. cerevisiae itself is [5].
More recently the use of oenological starter cultures not belonging to S. cerevisiae has been adopted. Often these species are inoculated with some overlap to a thriving S. cerevisiae population. These include “non-Saccharomyces” yeast (NSY), effectively any yeast not part of the Saccharomyces spp., as well as oenological lactic-acid bacteria (LAB). Given that the vast majority of wine producing nations are observant of OIV practices, commercialized cultured are required to be natural isolates derived from a winemaking environment [6].
The benefits by applying such cultures will vary on depending on the application, but predominantly these are used to enhance the overall sensory profile in some way, or for biological protection against spoilage processes. In the case of oenological bacterial cultures, their use could be for biological protection, although in the vast majority of cases these are inoculated in order to initiate malolactic fermentation (MLF).
1.1 Non-Saccharomyces yeast
Naturally present in the winemaking environment, NSY make up a significant part of the microbiome of a given fermenting wine, not just in musts where indigenous alcoholic fermentation is encouraged [7], but also in those inoculated with commercial S. cerevisiae starters. While in these “inoculated” fermentations the inoculated strain of S. cerevisiae proliferates, thereby giving the winemaker a more predictable outcome, other species are present to some degree at different stages of the process. In contrast, in “un-inoculated” fermentations, NSY species will be dominant in the early stages of fermentation [3]. The question of what effects specific these species can have, has led to them being isolated, then studied in a controlled manner. Having the potential to impart positive impacts upon a given wine has led to them being evaluated for their commercial appeal, which in turn led to the launch of the first NSY cultures around 20 years ago. Initial commercialized starters were a blend of NSY and S. cerevisiae, followed later by single-species starters. In the years since, the NSY species have been launched as commercial wine yeasts, includes but is not limited to; Torulaspora delbreuckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Metschnikowia fructicola, Pichia kluyveri and Hanseniaspora vineae, as detailed in Table 1.
Species
No. of products
Suppliers
Hanseniaspora vineae
1
Oenobrands
Lachancea thermotolerans
9
AEB, Chr. Hansen, Lamothe-Abiet, Laffort, Lallemand, ICV
AEB, Agrovin, Chr. Hansen, Lamothe-Abiet, Laffort, Lallemand, ICV
Table 1.
List of commercially available NSY cultures in France, 2022 [8].
The application of NSY species in winemaking has focused on two areas. The first of these is organoleptic impact. This could be from one of the following or a combination of; the production of volatile flavor compounds, the modulation of acidity or the production of non-volatile compounds that affect the perceived texture of a wine. In contrast, biological protection has been the other area where NSY have been studied and applied. This practice extends to applying NSY to prevent spoilage organisms from proliferating, to preventing oxidative reactions in must, or to ensuring certain desirable organisms have a healthy environment in which to flourish.
Typically, NSY is inoculated sequentially in winemaking, as represented in Figure 1. This allows the NSY population to dominate the winemaking environment, before S. cerevisiae is later introduced complete the alcoholic fermentation.
Figure 1.
The depiction of sequential inoculation with NSY. NSY is introduced to must first, its population shown by the solid blue line. After a period of 2–3 days, S. cerevisiae (dotted green line) is then inoculated, so that alcoholic fermentation can complete. The sugar concentration is shown by the dashed black line.
1.2 Oenological bacteria
Oenological bacterial cultures were developed in the later part of the 20th century. Then, as with today, the two species that were initially commercialized were Oenococcus oeni and Lactiplantibacillus plantarum. The former was developed as an alternative to relying on adventitious lactic-acid bacteria to develop in a wine, whereby MLF could be managed more effectively. The first “direct inoculation” starter for MLF, Viniflora® Oenos™, was launched in 1993. Its efficacy was based on implanting a high number of viable cells into a given wine, above the threshold of 1.0E+6 cfu/ml required for MLF to start [9]. In contrast, the first oenological culture based on L. plantarum was for a deacidification in juice or must, by completing a partial MLF at this early stage of winemaking.
The process of MLF was seen as being distinct from alcoholic fermentation, so inoculation of O. oeni starters generally took place after S. cerevisiae had finished converting sugars to ethanol. The only exception was the L. plantarum mentioned above, as this was designed to be inoculated prior to alcoholic fermentation. Nevertheless, whether before or after, the two fermentations of alcoholic and malolactic fermentations in wine were seen as being distinct. In the more recent past this approach had been challenged by the adoption of the practice known as “co-inoculation”. Although successfully demonstrated since 1985 [10], adopting the practice was initially very slow. Since the turn of the century, the practice has become more widespread. At the same time the risks associated with co-inoculation are better understood, and measures can be taken to minimize deviations. This can be done in two ways, firstly by ensuring the winemaking parameters such as pH, temperature and sulfite concentrations are conducive to both the yeast and bacteria being employed. Secondly, through selecting both yeast and bacterial strains that do not exhibit antagonism, risks of deviations are minimized [11]. The overlap of yeast and bacterial populations during co-inoculation are shown in Figure 2.
Figure 2.
The population overlap between S. cerevisiae and O. oeni when using co-inoculation, when compared to the more traditional sequential MLF. The relative population of S. cerevisiae is depicted in gray with a dashed outline, while O. oeni is depicted in blue with a solid outline.
A further, emerging application of oenological bacteria in winemaking is by utilizing L. plantarum for biological protection against spoilage organisms in musts. While the topic of biological protection is relatively recent, it was initially only focused on NSY [12]. It is apparent that there is a role for bacteria to play in this as well [13, 14]. This is highlighted in Figure 3, summarizing the “active” and “passive” mechanisms at play when using microbial cultures for biological protection.
Figure 3.
The passive and active strategies used when applying culture for biological protection, taken from [15].
In the nutrient and oxygen poor environment of a fermenting wine, by the end of the process, S. cerevisiae is recognized as becoming the dominant species of yeast. This extends to a wine fermentation where a NSY has been inoculated, although the population numbers S. cerevisiae can reach are generally lower than when compared with S. cerevisiae-only wines [16]. The converse effect has also been clearly demonstrated, where an active population of S. cerevisiae, can cause a significant decline in populations of NSY [17]. This may be due to competition for space, nutrients or through the production of inhibitory substances. Transcriptomics suggest that both S. cerevisiae and NSY strains impact upon the genetic expression of one another in a mixed fermentation [18].
Given that classical microbiological plating has been used for most of these population studies, it has been proposed more recently that NSY are not necessarily dead but could be in a viable but non-culturable state (VBNC). While these population can later be revived [19], it does not detract from the dominance seen from S. cerevisiae. It merely challenges the correct state of any NSY populations that could be present.
Like NSY, oenological populations of LAB can also be impacted by the presence of S. cerevisiae. The causes for this will be examined in more detail in the following sections. Conversely, S. cerevisiae populations are seldom impacted by inoculated LAB. This is unsurprising given the MLF still takes place after alcoholic fermentations for many wines, but even when co-inoculation practices care used, there has been little impact demonstrated on S. cerevisiae populations [20, 21].
2.1.1 Cell to cell contact
Physical contact between cells from differing yeast populations (cell-cell contact) has been demonstrated as an important aspect when assessing population dynamics during winemaking. Investigated by using double-compartment fermentation vessels (P. kluyveri), or by sterile-filtration between inoculating yeast populations (L. thermotolerans, T. delbrueckii), S. cerevisiae can become increasingly dominant over NSY when physically together [17, 22]. It has been demonstrated that this phenomenon is most associated with the presence of the FLO family genes in S. cerevisiae, which control flocculation [23].
Some studies have also showed that by physically removing NSY, the health of S. cerevisiae is subsequently affected. This most likely due to removing nutrients along with the NSY, rather than any absence of cell-cell contact [24, 25, 26].
2.2 Nutrient requirements
Wine microorganisms require nutrients to survive and thrive, so that the ultimate end-goal of producing a sound wine in a timely manner can be realized. Given that the wine microbiome changes during fermentation, whether with inoculated or native organisms, the potential for competition for nutrients exists throughout. It is clear however the requirements for different species, or even different strains within a species, will vary. In order to properly manage wine fermentations, these differences need to be considered and wherever possible be managed.
2.2.1 Assimilable nitrogen
Nitrogenous compounds, typically amino acids and ammonium ions are required by yeasts for growth. The sum of the concentrations of these are expressed as Yeast Assimilable Nitrogen (YAN). The nitrogen demand between different strains S. cerevisiae does vary [27], however the typical guideline for a healthy alcoholic fermentation in wine is a YAN concentration > 150 mg/L. In contrast, oenological LAB cannot utilize ammonia, with the requirement for a healthy population of O. oeni being 60 mg/L of amino acid nitrogen [28]. The inclusion of additional microbial species, sometime in the winemaking process, can thereby lead to competition with a higher demand for YAN when NSY and S. cerevisiae populations are together [29].
The nitrogen requirements of NSY will also vary between species [30]. When studying commercial strains of L. thermotolerans, T. delbrueckii, P. kluyveri and M. pulcherrima, it was shown that the first two species consume the available nitrogen, both amino acids and ammonium, at a much faster rate than the latter two. Furthermore, upon filtering samples inoculated with these two species, then inoculated with S. cerevisiae, primary fermentation was unable to complete. In contrast, when the same conditions were applied to both P. kluyveri and M. pulcherrima, there was much less competition for nutrients, thereby allowing primary fermentation to finish. In all cases the fermentation completed when S. cerevisiae was inoculated without filtration [25]. This suggests lysing of the NSY cells is required in order to supply enough nitrogen to the S. cerevisiae. A further facet examined in the same study is around the release of certain amino acids back into the fermenting wine by P. kluyveri and M. pulcherrima early in the first 48 hours of winemaking, as detailed in Figure 4. While limited scientific data exists on this, it does propose a mechanism for certain strains of NSY to be used in low-intervention winemaking, to help ensure native S. cerevisiae is adequately fed.
Figure 4.
The uptake and subsequent release of amino acids by S. cerevisiae and four commercial NSY in the first 48 hours of fermentation. In the cases of the P. kluyveri and M. pulcherrima there is significant release of certain amino acids back into the fermenting wine. Taken from [25].
When it comes to oenological bacteria, there is also the risk of competition between S. cerevisiae and the bacteria being utilized. In most cases, bacteria are inoculated after yeasts are, and will have a lower nitrogen demand. Given that MLF is seen as requiring an amino nitrogen concentration of 60 mg/L, depletion of nitrogen is generally only seen against bacteria, from yeasts, not the other way around. Given that the concentration of assimilable nitrogen in a wine pre-MLF can vary, including as a result of differing YAN requirements between strains of S. cerevisiae, to ensure successful MLF it is often recommended to measure the amino nitrogen concentration, and address any nitrogen deficiencies, at the start of this process.
2.2.2 Thiamine and B1 vitaminers
Thiamine is necessary for successful growth of wine yeasts. While many yeasts can synthesis this compound, it has been suggested that it is not possible under the stressful conditions of a wine fermentation. Previous work has suggested that indigenous strains of Hanseniaspora uvarum can quickly deplete exogenous thiamine present in must, thereby depriving S. cerevisiae of this vitamin and leading to stuck alcoholic fermentation [31]. More recent work investigating another apiculated yeast, H. vineae, found no such effect on the S. cerevisiae present [32]. The Thiamine uptake of T. delbreuckii has also been investigated and found to be consumed at roughly a similar rate as S. cerevisiae [24]. Three other species Starmerella bacillaris, M. pulcherrima and T. delbrueckii demonstrated slower uptake of Thiamine and other B1 Vitamers at a lower rate than S. cerevisiae [33, 34].
2.2.3 Pantothenic acid and B5 vitaminers
The uptake and release of Pantothenic acid, and its B5 cofactor form Coenzyme A by NSY has been investigated. All strains studied in [32] found that both B5 vitaminers were synthesized early after inoculation, however T. delbrueckii was found to re-consume a proportion. This has implications when later inoculating S. cerevisiae, as competition for these vitamins could potentially lead to decreased numbers and sluggish fermentation. In contrast, by synthesizing B5 vitaminers, S. bacillaris and M. pulcherrima could provide a beneficial environment for a later S. cerevisiae inoculation.
2.3 Inhibitory compounds
A range of inhibitory substances can be produced by oenological organisms, whether by S. cerevisiae, NSY or by LAB. For the latter, inhibitory compounds have not been reported with commercialized starters, but sluggish fermentation has been linked to adventitious spoilage strains of LAB [35]. Yeasts however are capable of producing a range of toxic compounds, the most obvious of which is ethanol. In fact the production of ethanol, and the subsequent toxicity to NSY is seen as one of the key mechanisms for S. cerevisiae to dominate a wine fermentation.
2.3.1 Sulfur dioxide
While sulfur dioxide (SO2) remains the preeminent preservative used in winemaking, to control both oxidative and microbial spoilage, it is also a compound that can be synthesized by S. cerevisiae by the sulfate assimilation pathway. The concentrations of SO2 produced depends on both the strain, and the winemaking conditions, and can vary widely. When expressed as Total-SO2 (the sum of the concentrations of molecular SO2, free bisulfate ions and bisulfate ions bound to other moieties) the concentration produced can range from 20 to 200 mg/L, [36]. This has very important implications for when applying NSY or LAB con-currently as these two families have a much lower tolerance to SO2 than S. cerevisiae, or at least those strains of S. cerevisiae selected as oenological starters. This therefore reinforces the typically applied sequential inoculation of NSY, allowing them to proliferate before S. cerevisiae comes to dominate. For bacteria, to ensure successful MLF, it is therefore suggested to ensure the S. cerevisiae employed for the alcoholic fermentation does not produce excessive SO2. A good rule of thumb is to keep the concentration of T- SO2 below 40 mg/L, although specific tolerances can be cross-referenced with the supplier of MLF bacteria.
2.3.2 Medium chain fatty acids
Octanoic, decanoic and dodecanoic acids (C8, C10 and C12 respectively) are medium chain fatty acids (MCFA), always produced by wine yeasts to some degree during wine fermentation. In fact, ethyl esters of these compounds are seen as beneficial flavor compounds in wine while higher concentrations of free MCFA are seen as having a direct effect of sluggish fermentation [37]. While the toxicity of MCFA to NSY is not well studied, the production of these has been investigated. Oxygenation of mixed fermentations, with either Lactobacillus L. thermotolerans or T. delbrueckii and S. cerevisiae, gave lower MCFA concentrations than the same combinations fermented anaerobically, as well as the S. cerevisiae-only control. Low MCFA concentrations were also seen in single-strain NSY fermentations. This suggests that both of these commercial NSY strains involved in the study will naturally produce lower concentrations of MCFA [38]. These results also lead the authors to conclude with the presence of oxygen encouraged the incorporation of MCFA into longer fatty acids via Acetyl-CoA carboxylase activity.
MCFA are inhibitory to oenological bacteria, especially in their unesterified free forms. As with SO2, it is important that that the concentration of these is kept below a certain threshold. This is below 12.5 mg/L for C10 and 2.5 mg/L for C12 [39], although individual tolerances between different strains of LAB will vary. Fermenting S. cerevisiae at warmer temperatures had been shown to give lower MCFA concentrations in the final wine [40], however the innovative approach of utilizing NSY combined with oxygenation could be another approach.
2.3.3 Killer toxins
The ability of yeast strains to both produce specific extracellular proteinaceous compounds that can kill other yeasts, referred to as killer toxins, has been well studied. For S. cerevisiae there are three killer toxins, referred to as; K1, K2 and K28, and these are only active within the species. Strains can either produce a killer toxin or be sensitive to toxins. Within oenology, the killer toxin status between different strains of commercial S. cerevisiae starters is therefore often published by yeast manufacturers, and expressed as ‘positive’, ‘sensitive’ or ‘neutral’. The latter referring to strains where no toxin is produced and there is not sensitivity to the three known killer factors for S. cerevisiae.
Killer toxins have also been shown to be produced by various NSY strains, and utilizing these has been proposed as a biological protection strategy in winemaking. In [41] only a minority of these are active at wine pH, and when they are, none have been shown to be from NSY species that have been commercialized. T. delbrueckii is capable of producing a killer toxin referred to at TDKT, however no effect on commercial S. cerevisiae strains from this toxin has been observed [42]. Likewise, no inhibition of S. cerevisiae from killer toxin was seen in when investigating a commercial strain of T. delbreuckii in [24].
A similar situation exists for L. thermotolerans, with some studies suggesting strains of this species are positive for a killer toxin against S. cerevisiae. As the methodology for establishing such killer factors is carried out at pH 4.50, higher than that of wine, it can be argued that it is of limited value in oenology. Conversely multiple studies with L. thermotolerans in combinations with various strains of S. cerevisiae, repeatedly demonstrate the dominance of the latter species [43].
P. kluyveri has long been associated with producing killer toxin [44] active against S. cerevisiae, more recently labeled as PKKP [45]. While exhibiting less activity at the relatively low pH range of wine, the question of whether commercialized strains of this species produce this toxin should naturally be raised. It would seem unlikely, and while not directly studied in [22], it would appear that no killer toxin from the commercial strain of P. kluyveri used (Viniflora® FrootZen™) affected the growth of the S. cerevisiae employed.
M. pulcherrima has also been found to be positive for killer toxin against a sensitive strain of S. cerevisiae in one study [46], however the same arguments around the validity of the test being carried out at high pH apply. Interestingly, two of the authors conclude in a later paper that much of the antimicrobial activity of this species is via the production of pulcherriminic acid rather than killer factor [47]. In the same later study and using the same test for killer toxin, no inhibition was seen against the 18 commercial strains of S. cerevisiae tested.
2.3.4 Other biological inhibitors
Beyond killer toxins, there are other peptide-based inhibitory substances produced in mixed fermentations. Anti-microbial activity was seen against L. thermotolerans and T. delbrueckii when investigating a secreted protein fraction, 2-10 kDa in size, from a specific S. cerevisiae strain [48], later shown to be derive from a specific protein, abbreviated as GADPH [49].
Extracellular vesicles (EV) are another potential group of potentially inhibitory compounds in yeast-yeast interactions. EV’s 100–200 nm in size were found to be produced by all six species studied in [50]; S. cerevisiae, L. thermotolerans, H. uvarum, Candida sake, M. pulcherrima and T. delbrueckii. While in this study inhibition of the T. delbrueckii toward S. cerevisiae was seen, it was not from the most abundant EV produced. No doubt more light will be shed on this topic in the coming years.
While killer toxins are seen as a yeast-yeast interaction, there is also evidence that yeast derived peptides can be inhibitory to oenological bacteria. For instance, the protein fraction derived from GADPH and studied in [49] was also found to be inhibitory toward a strain of O. oeni. Other studies where anti-microbial peptides derived from S. cerevisiae were found to inhibit bacteria suggested differing sizes of these substances. This suggests the peptides themselves, and their production, varies across the species [51].
With the goal of producing expressive, sound and fault-free wines in an economic fashion, cultures of species other than S. cerevisiae, whether LAB or NSY have become an indispensable tool at the winemaker’s disposal. Whether utilized to ensure timely fermentation processes, to mitigate against spoilage, or to optimize the resultant organoleptic characters, it is expected that in most cases there will be an impact on the final wines composition from these species.
3.1 Mitigation of spoilage characters through biological protection
3.1.1 Ethyl phenols
The ethyl phenols 4-Ethyl Guaiacol (4-EG) and 4-Ethyl Phenol (4-EP) can arise in wine due to the presence one of the most serious of wine contaminants, Brettanomyces bruxellensis. While SO2 has traditionally been used to help mitigate against the outgrowth of B. bruxellensis, this approach is likely to become more challenging in the future due to increasing growing season temperatures leading to increased wine pH, and the ability of B. bruxellensis to adapt to SO2 [52].
Two bioprotective strategies can be employed against B. bruxellensis. The first is to treat must by inoculation with a pre-fermentative NSY, early in the winemaking process. Some strains of M. pulcherrima have been shown to have antimicrobial activity against B. bruxellensis [47]. The mechanism for this has been demonstrated to be due to the chelation of iron by pulcherriminic acid, thereby starving the contaminants of this essential mineral. Interestingly, this mechanism seems to have little effect on S. cerevisiae. Ethyl phenol development from B. bruxellensis contamination does however generally arise later in the winemaking process, after alcoholic fermentation is complete. Whether this early inhibition of B. bruxellensis persists is inconclusive.
The second and most effective microbial strategy against B. bruxellensis that can be utilized is to inoculate wine, or in the case of co-inoculation must, with MLF bacteria. Given that B. bruxellensis develops later in the winemaking process, by ensuring MLF starts and finishes in a timely manner from the inoculated bacteria, the opportunity for this species to build numbers is severely limited. In contrast, while waiting for adequate numbers of O. oeni to develop when relying on adventitious populations, B. bruxellensis has the opportunity to grow in the warm environment, largely free of sulfites. While the main protective effect seen is simply from the ability to microbially stablise a given wine sooner, this has been recent evidence to suggest O. oeni exhibits a fungistatic effect on B. bruxellensis, through cell-cell interactions. This is however, likely to be strain specific [53].
3.1.2 Volatile acidity
The excessive production of volatile acids in wine, predominantly acetic acid (AA) is a serious deviation, and limits of such acids are often mandated by law. While AA can be caused by spoilage organisms, particularly apiculated yeast and Acetic Acid Bacteria, it can also arise from the population of S. cerevisiae present. During alcoholic fermentation there will always be a small quantity of AA produced, so ensuring this stays at a low concentration is important. The tendency of S. cerevisiae to produce AA increases with more osmotic stress. Specific NSY strains of L. thermotolerans, T. delbrueckii, S. bacillaris and M. pulcherrima in comparison have often been shown to produce less AA in a single-species fermentation [46] however the lower AA effect also occurs in mixed NSY-S. cerevisiae fermentations with the same strains. This suggested commercial strains of NSY are an effective tool in the styles of wine most susceptible to elevated AA, for instance wines from very high-sugar musts [54].
When it comes to AA production from spoilage organisms, often oxygen is required to this to occur. Excluding oxygen during the winemaking process therefore become an important measure to limit AA production. While little scientific data exists on oxygen-uptake rates of NSY c.f. S. cerevisiae, this is an emerging area for further study [55].
3.1.3 Chemical oxidation
Chemical oxidation in winemaking is generally prevented by good cellar practices, that prevent oxygen exposure, combined with the use of anti-oxidants, predominantly SO2 and to a much lesser extent ascorbic acid. Like S. cerevisiae, NSY will readily consume dissolved oxygen in must. Identifying candidates that do this most rapidly could have application in low-sulfite winemaking. This could be combined with the properties of M. pulcherrima to produce pulcherriminic acid, a substance which will chelate with iron ions [47]. Given many oxidative reactions in must and wine are catalyzed by the presence of metal ions, effectively removing these from the must/wine matrix could prevent potentially mitigate against oxidation to some degree [56].
3.1.4 Biogenic amines
Biogenic amines (BA) arise from the decarboxylation of amino acids by certain wine microorganisms. Histamine is the most predominant BA found in wine, arising from the decarboxylation of histidine, but others produced include cadaverine, putrescine and tyramine. While BA primarily arise from LAB, yeast species can also play a role. To limit their formation, it is therefore suggested to use commercial strains with little or no decarboxylase activity against histidine and the other relevant amino acids [57]. Given BA arise from the activity of adventitious flora, the use of strains for biological-protection can potentially also play a role.
3.2 Flavor compounds
In wines fermented in conjunction with species other than S. cerevisiae, the resultant flavor compounds produced have a significant effect on the final sensory profile. These effects have been extensively studied in both the scientific literature and winemaking field trials. The following is by no means an exhaustive list of such work, but is a summary of the most important compounds, from a wine-aroma point of view.
3.2.1 Esters
Esters are compounds formed in wine by the combination of acids and alcohols, either by the action of microorganisms such as yeasts or bacteria (biological esterification) or during storage and aging (chemical esterification). They are produced by yeast via esterase activity, and may be volatile or non-volatile.
A wide range of yeast-derived esters are formed during a wine fermentation. This includes ethyl esters, acetates and esterified forms of fatty acids and higher alcohols. An increase in the concentrations of the sum of all esters measured was seen in [58, 59], where sequential mixed inoculations of S. cerevisiae and NSY were evaluated against S. cerevisiae only controls. In the former paper L. thermotolerans, T. delbrueckii and H. vineae were studied, whereas three strains each of L. thermotolerans, Metschnikowia spp. and S. bacillaris were the NSY species investigated in [59].
One particularly important ethyl ester to consider is ethyl lactate (EL), which is known to impart a strawberry-like aroma. While S. cerevisiae-only wine fermentations generally have very low concentrations, L. thermotolerans is able to produce L-lactic acid from grape sugars, which in turn acts as a precursor for EL production [43]. EL production is not however restricted to L. thermotolerans as LAB will also produce L-lactic acid, and EL. In the case of L. plantarum however, although somewhat strain dependent, the production of EL can far exceed that from O. oeni. L. plantarum therefore has the potential to make a positive contribution to wine aroma [59].
Acetate esters can often be more readily synthesized by NSY when compared to S. cerevisiae. In some cases this can be beneficial, such as for 2-phenylethyl acetate, which imparts a pleasant rose-like aroma. H. vineae in particular has been selected for this characteristic [60], although it has also been measured at more moderate concentrations in fermentations with T. delbrueckii also [61].
Isoamyl acetate is another acetate ester that has been widely studied. Giving a banana-like aroma, some strains of S. cerevisiae are promoted on the ability to produce this compound, using isoamyl alcohol as a precursor. While concentrations of this compound produced with respect to S. cerevisiae varies across studies, it is generally observed that P. kluyveri will increase concentrations [25].
While isoamyl acetate and 2-phenylethyl acetate are important acetate esters, without negative connotations, production of acetate esters may not always be beneficial to wine quality. One example is ethyl acetate, which while contributing positively at low concentrations, will give solvent-like characters at elevated levels. While some strains of H. uvarum are capable of producing very high concentrations [62], fortunately commercial NSY do not produce elevated concentrations of ethyl acetate. In fact, often lower ethyl acetate concentrations are measure in wine co-fermented with both NSY and S. cerevisiae compared to S. cerevisiae alone [61, 63].
Ethyl esters of MCFA such as ethyl hexanoate, ethyl octanoate and ethyl decanoate also play an important role in wine flavor chemistry. Research shows that co-fermenting with NSY does influence the concentration of these [46]. Whether concentrations increase or decrease for a particular ethyl ester of a MCFA depends very much on both species and strain, as well as the S. cerevisiae being employed.
3.2.2 Thiols
Three specific sulfur-containing flavor compounds, 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP) have been extensively investigated for the positive effects that they have on wine aroma. While imparting aromas of boxtree, passionfruit and grapefruit these are in contrast to much smaller mercaptans, such a dimethyl disulfide and methanethiol, which impart negative characters. As the three volatile thiols are not naturally present in wine grapes, yeasts are required to release these from their precursors, cysteinylated and glutathionylated forms of 4MMP and 3MH. Further conversing of 3MH to 3MHA then takes place from alcohol-acetyltransferases.
While many S. cerevisiae starters are marketed for their ability to release volatile thiols, NSY can play important role too. P. kluyveri has been investigated, and subsequently a commercialized NSY starter has been promoted around increasing 3MH and 3MHA concentrations. Interestingly, the interaction between this species and the strain of S. cerevisiae used is crucial. While the combination of P. kluyveri and S. cerevisiae can lead to an increase in both 3MH and 3MHA, for some strains of S. cerevisiae this led to an increase in only one of these, or in some cases neither [64].
Other NSY species shown to increase volatile thiol concentrations include T. delbreuckii, L. thermotolerans, M. pulcherrima and S. bacillaris [64, 65].
3.2.3 Terpenoids
Terpenoids such as monoterpenes, sesquiterpenes and norisoprenoids are an important group of aroma compounds in wine. Generally existing primarily in non-volatile “bound” forms in grapes, the free equivalents are released by glycosidase activity. An increase in overall total terpene concentrations from co-fermentations of NSY and S. cerevisiae has been demonstrated in various studies, which is unsurprising given NSY exhibit greater β-glucosidase activity when compared to S. cerevisiae [66]. This effect however is not seen in all cases, suggested that under winemaking conditions, some strains of S. cerevisiae have roughly equivalent activities [63]. Interestingly O. oeni can also have significant glycosidase activity, resulting in increased concentrations of free terpenoids during MLF [67].
3.2.4 Others
The production of higher alcohols has shown to vary across yeast species, which can vary further whether co-fermented with S. cerevisiae. This can be linked to some degree to amino acid consumption, with production via the Ehrlich pathway, but not exclusively so [25].
2,3 Butanedione, commonly referred to as diacetyl, as another biologically important flavor compound. Its production in wine derives from citrate-fermentation by LAB, primarily O. oeni, followed by chemical oxidation of the intermediate compound, α-acetolactate. Co-fermentation between O. oeni and S. cerevisiae will have a significant reduction on concentrations of diacetyl formed, both through viable yeast cells being able to take up this compound, and by the presence of an active population of S. cerevisiae ensuring oxygen concentrations are depleted.
3.3 Acidity modulation
In winemaking, the biological process which has the largest impact upon a wine’s acidity in MLF. When carrying out this process with O. oeni in conjunction with alcoholic fermentation, some studies have shown a slight increase in pH or titratable acidity, compared to running both fermentations sequentially [21]. The bacterial genus that offers the most oenological promise for carrying out such acidification however is Lactobacillus, utilizing the production of lactate from grape sugars. Due to the generally low tolerance to ethanol from Lactobacilli, this process needs to take place early in the winemaking process, so most likely with an overlap with the presence of S. cerevisiae.
One particular species of NSY that offers an alternative to LAB for MLF is Schizosaccharomyces pombe, through maloalcoholic fermentation [68]. While two examples have been commercialized, their use to date has been very limited.
L. thermotolerans is the most high-profile NSY species investigated for the effect that it can have on wine acidity. Like Lactobacillus spp., it can synthesize lactate from sugars. The immediate application for such yeasts is for musts lacking in acidity [43], so is not surprising that it is one of the most utilized NSY species in winemaking [8], producing up to 8 g/L of Lactic acid. While helping to balance low acidity, further benefits include a potential shift to higher concentrations of molecular SO2 from unbound sulfites, as well as potentially lower final ethanol concentrations. Both are depicted in Figure 5.
Figure 5.
The effect on lactic acid production (green line), alcohol content (blue line), pH, and molecular SO2 when running sequential fermentation, first with L. thermotolerans (green yeast) and then with S. cerevisiae (pink yeast). Taken from [12].
3.4 Textural elements
A long-held belief among some parts of the winemaking community is that wines fermented using spontaneous fermentation, so by inference using adventitious NSY to a large degree, give a fuller mouthfeel than ones simply inoculated with S. cerevisiae. Research into mixed fermentations of NSY and S. cerevisiae has been able to clearly demonstrate a connection between a more diverse yeast ecology and increased palate-weight, with NSY often producing levels of polysaccharide well in excess of S. cerevisiae alone [46], as detailed in Table 2. It is unsurprising that yeast cell walls are largely composed of polysaccharides, which can be released into the medium upon cell lysis. Polysaccharides are compounds soluble in alcohol, and the combination of polysaccharides with tannins and anthocyanins increases viscosity and fullness in the mouth, provides complexity and aromatic persistence, reduces astringency, thus improving the structure, density and texture of the wine and stabilizes the coloring matter [69]. Although increases have been observed with many species of NSY, T. delbrueckii is seen as the species which can give the largest increase to the final polysaccharide concentration of a given wine [70, 71].
Inoculum, (cells ml–1)
Ethanol (% v/v)
pH
Total acidity (g l–1)
Volatile acidity (g l–1)
Glycerol (g l–1)
Δ Polysaccharides (mg l–1)
S. cerevisiae 107
13.93 ± 0.06a
3.20 ± 0.04a
7.05 ± 0.04a
0.46 ± 0.01a
6.23 ± 0.54a
97 ± 10a
S. cerevisiae 105
13.87 ± 0.00a
3.16 ± 0.05a
7.12 ± 0.11a
0.47 ± 0.01a
6.65 ± 0.05a
98 ± 11a
S. cerevisiae 103
13.88 ± 0.03a
3.17 ± 0.21a
7.02 ± 0.05a
0.50 ± 0.06a
6.46 ± 0.52a
68 ± 3.0a
C. zemplinina + S. cerevisiae 107
13.83 ± 0.04a
3.21 ± 0.01a
6.88 ± 0.27a
0.43 ± 0.04a
6.25 ± 0.30a
123 ± 42a
C. zemplinina + S. cerevisiae 105
13.78 ± 0.05a
3.15 ± 0.05a
6.84 ± 0.02a
0.44 ± 0.06a
7.18 ± 1.30b
140 ± 42a
C. zemplinina + S. cerevisiae 103
13.64 ± 0.04b
3.08 ± 0.18b
6.88 ± 0.04a
0.52 ± 0.01a
7.95 ± 1.28b
181 ± 48a
L. thermotolerans + S. cerevisiae 107
13.80 ± 0.02a
3.16 ± 0.01a
7.30 ± 0.07a
0.38 ± 0.01b
6.95 ± 0.20b
133 ± 1.0a
L. thermotolerans + S. cerevisiae 105
13.80 ± 0.01a
2.97 ± 0.03b
9.00 ± 1.96b
0.40 ± 0.00a,b
7.29 ± 0.96b
139 ± 10a
L. thermotolerans + S. cerevisiae 103
13.70 ± 0.18a
2.90 ± 0.01b
9.20 ± 1.93b
0.40 ± 0.00a,b
7.58 ± 0.46b
158 ± 3.0a
T. delbrueckii + S. cerevisiae 107
13.90 ± 0.04a
3.19 ± 0.01a
7.12 ± 0.02a
0.38 ± 0.01b
5.88 ± 0.04a
157 ± 16a
T. delbrueckii + S. cerevisiae 105
13.85 ± 0.08a
3.10 ± 0.08a,b
7.36 ± 0.5la
0.40 ± 0.04a,b
6.14 ± 0.22a
269 ± 44b
T. delbrueckii + S. cerevisiae 103
13.76 ± 0.04
3.08 ± 0.11a,b
7.34 ± 0.49a
0.41 ± 0.01a,b
6.29 ± 0.61a
308 ± 42b
M. pulcherrima + S. cerevisiae 107
13.87 ± 0.01a
3.40 ± 0.08c
6.33 ± 0.27a
0.30 ± 0.04b
6.53 ± 0.27a
120 ± 10a
M. pulcherrima + S. cerevisiae 105
13.79 ± 0.13a
3.39 ± 0.14c
6.50 ± 0.16a
0.34 ± 0.07b
6.98 ± 0.00b
126 ± 10a
M. pulcherrima + S. cerevisiae 103
13.65 ± 0.19b
3.40 ± 0.00c
6.64 ± 0.37a
0.33 ± 0.01b
7.25 ± 0.25b
154 ± 17a
Table 2.
Analytical profiles of wines fermented as mixed culture of S. cerevisiae and four different NSY. While an increase in polysaccharide concentration is measured for all four NSY species, the increase is significantly greater for T. delbrueckii. Taken from [46].
Data are means ± standard deviations of two independent experiments. Values displaying different superscript letters (a, b, c) within each column are significantly different according to the Duncan test (0.05%).
The microbiome of grapes, must and wine during winemaking is complex, with multiple interactions taking place between populations, whether naturally present or inoculated. While S. cerevisiae is dominant at the later stages of alcoholic fermentation, there are key interactions between this and other microbial species, whether NSY or bacteria.
The use of oenological bacterial cultures and NSY has grown significantly in recent years, as winemakers strive to make expressive and fault-free wines, which are true to their origins. This could be to bring biological protection, enhance the organoleptic profile, or in the case of oenological bacteria, to induce MLF. In order to ensure success however, it is important to understand the potential relationships between inoculated populations.
The benefits of utilizing species other than S. cerevisiae are multiple, whether increasing volatile flavor compounds, helping to prevent the effects of spoilage or impacting the acidic profile of the final wine.
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Written By
Duncan Hamm and Bernardo Muñoz González
Submitted: 21 September 2023Reviewed: 21 September 2023Published: 16 November 2023
Open access peer-reviewed chapter
