Application of Wine Yeast Starter Cultures in the Production of Grape and Fruit Wines

Abstract

Significant advances in winemaking equipment and processes, as well as a deeper understanding of the role of yeast, have significantly improved wine quality throughout history. This paper examines critical aspects related to the use of commercial wine yeast starter cultures in the fermentation of grape and fruit wines, with a focus on berry wines and blackberry wine, which is the most predominant berry wine in Croatia. While the production of grape wines remains the most significant, fruit wines are gaining importance due to their composition, which contains a variety of bioactive compounds. Although spontaneous fermentation is still preferred by some winemakers, controlled or inoculated fermentation, based on the use of wine yeast starter cultures, is predominantly employed in modern winemaking. The selection of suitable yeast strains for grape wines is easier than for fruit wines, as the broader availability of commercial yeasts for grape wines contrasts with the limited selection offered for fruit wine production due to the smaller fruit wine market. The selection of Saccharomyces and, more recently, non-Saccharomyces yeast strains with desirable characteristics are crucial for the production of high-quality wines. Selection criteria for wine yeasts have evolved to meet modern consumer preferences and focus on technological properties, secondary flavor development and health effects.

1. Introduction

Since the beginning of written history, there has been evidence that humans have produced fermented beverages, although the role of yeast in alcoholic fermentation was unknown. While researching wine and beer in the mid-19th century, Louis Pasteur demonstrated that yeasts found on the surface of grapes are responsible for spontaneous fermentation and proved that this process produces not only ethanol and CO₂ but also secondary products of yeast metabolism, which are now known to play a role in aroma formation [1]. Since then, numerous microbiological, biochemical, molecular biological and genetic studies have been carried out, which have contributed to the expansion of knowledge about wine yeasts and their role in winemaking. Building on this knowledge, the selection of wine yeasts has been conducted for many years, leading to the development of yeast starter cultures for use in modern winemaking [2]. Initially, the primary selection criterion was the production of low concentrations of undesirable compounds. However, contemporary selection criteria are significantly more stringent and vary depending on the type of wine. A fast, robust, and complete fermentation of sugars in must and fruit juices, resulting in a high alcohol content (>8% v/v) and a desirable aromatic profile, is the fundamental requirement for wine yeast [3]. Furthermore, the yeast must be tolerant to the presence of sulfur (IV) oxide added to must/fruit juice (for its antioxidant and antimicrobial roles), it must have the ability to produce a minimal amount of foam during fermentation and it should be quickly separated from the wine by sedimentation at the end of fermentation [3].

The development of starter cultures, which enable the production of high-quality wine, is important, as the wine market generates substantial income for the countries that dominate global wine production. The cultivation of grapes, which spread throughout Europe with the expansion of the Roman Empire, has played a central role in European culture for centuries. The European Union is the world’s largest producer (over 47%), consumer (48%), and exporter of wine. Eurostat reports that in 2020, there were 3.2 million hectares of vineyards, which represents 2% of the EU’s utilized agricultural land and 45% of the global vine-growing area [4]. In Croatia, wine production is traditionally important both in households and in industry and represents great potential for the future.

In addition to the traditional production of wine from grapes, the potential for the production of fruit wines should not be overlooked. Rapid transformation in the wine world occurred recently and was linked to changes in consumers’ preferences, consumption habits, climate, new regulations, and economic resources. New consumers are attracted by new and innovative low-alcohol beverages with pleasant aromas and naturally enriched in bioactive compounds, attributes that are inherently characteristic of fruit wines [5]. Fruit wines are all wines obtained through the alcoholic fermentation of fruit juices other than grapes. Even though they have been homemade since the dawn of civilization, they are less well known than grape wines because their industrial-scale production has not reached the same magnitude. However, the significant health benefits of various fruits, including berries, have contributed to their growing market importance. The types of fruit wines produced, as well as their consumption, are shaped by regional geography and local fruit varieties, with the central European, or transitional, type of climate favoring wines made from apples, berries and sour cherries [6]. Generally, apple cider is the most predominant fruit wine on the EU market. However, this review will place more attention on berry wines, owing to their rich phenolic content, high antioxidant capacity, and unique flavor profiles, as well as the growing consumer interest in beverages rich in bioactive compounds with documented functional and health-promoting properties [7,8,9]. In Croatia, blackberry wine production is particularly noteworthy due to its traditional roots (especially in the continental regions), along with its market availability and constant consumer demand. Its distinctive sensory profile and chemical composition are why it has traditionally been used both as a dessert wine and as a remedy for anemia and insomnia [10]. Given all the aforementioned factors, this review paper will specifically tackle the use of starter cultures in the context of Croatian blackberry wines.

Considering the scale of fruit production in the EU and the fact that 20–30% of fruit is subject to postharvest losses, including surplus (e.g., from retail) or second-class fruit unsuitable for direct consumption, fruit wine production offers a sustainable approach for utilizing these raw materials. This process yields a product with enhanced added value, maintaining high quality despite the use of raw materials that would otherwise be discarded as waste while simultaneously contributing to waste reduction [11].

From a technological perspective, the processes for producing grape wines and fruit wines are quite similar. The primary differences lie in the composition and the preparation or adjustment of raw materials (fruits at the appropriate stage of ripeness), as many fruit varieties do not possess the optimal sugar-to-acid ratio for wine production, unlike grapes, which naturally have a high sugar content and low acid content. Furthermore, the essential nutrients needed for yeast growth are missing in many fruits. Therefore, fruit juices must be adjusted accordingly to make them suitable for winemaking [12]. Established grape wine technology and equipment can be easily adapted for the production of fruit wines.

This paper provides an overview of the development of yeast starter cultures and explores the key factors associated with the use of commercial wine yeasts in the fermentation of grape and fruit wines.

2. Spontaneous vs. Controlled (Inoculated) Fermentation: The Crucial Role of Yeast

Spontaneous fermentation (also called natural fermentation, uninoculated fermentation or fermentation with autochthonous flora) has traditionally been carried out in winemaking for thousands of years through the combined action of different yeast species that are part of the microflora of grapes (and fruits other than grapes) and the microbiota of the winery and grow in succession during must fermentation. At the beginning of vinification, non-Saccharomyces yeasts predominate and initiate the fermentation of the grape juice. Two to three days after the start of fermentation, ethanol, in a concentration of around 4% (v/v), inhibits their growth. After that, the strains of the genus Saccharomyces predominate and complete the alcoholic fermentation [13].

Controlling the winemaking process and the quality of the final product (i.e., wine or fruit wine) are important for producers, suppliers and end consumers. Based on the understanding of spontaneous fermentation, the concept of controlled fermentation emerged. This process is achieved by inoculating appropriate starter cultures of wine yeasts at concentrations that suppress the growth of undesirable wild yeasts. In practice, this fermentation method is referred to by various terms, including controlled fermentation, inoculated fermentation, directed fermentation, and guided fermentation [13,14]. In addition, the fermentation temperature is carefully controlled and maintained within the optimum defined parameters, since temperature influences various physiological and biochemical aspects of yeast, including their sensitivity to alcohol concentration, growth dynamics, fermentation rate, viability, and duration of the lag phase, as well as enzyme activity and membrane functionality [15].

Controlled fermentation has led to significant changes in the technology of wine and fruit wine production, resulting in better quality wines, especially in terms of sensory quality and chemical composition of the wine, with special emphasis on aroma and bioactive compounds (e.g., polyphenols) [16]. In contrast, spontaneous (uncontrolled) fermentation carries a higher risk of stuck or slow (sluggish) fermentation, contamination with spoilage microorganisms, and the development of less favorable aroma profiles [17]. Controlled fermentation ensures reliable fermentation kinetics and reproducibility of the fermentation process [18] while helping to avoid issues such as stuck or slow (sluggish) fermentation [17,19].

Under controlled conditions, fermentation lasts somewhat longer but is more uniform, sugar utilization is superior (more alcohol, less volatile acids), and the wine contains more CO₂ (fresher taste) and SO₂ (the wine is better protected from spoilage) [12]. In addition, the wines are clearer, have a more pronounced taste, aroma and varietal characteristics, and are more stable because the precipitation of tartar or tartrate is prevented during the appropriate course of the fermentation process [20].

Despite the well-documented advantages of controlled fermentation, some producers have expressed concerns regarding the excessive standardization (uniformity) of wine characteristics and flavors resulting from the use of commercial starter cultures of selected wine yeasts. Consequently, renewed interest has emerged in the application of autochthonous yeasts in winemaking, including spontaneous fermentation or the use of autochthonous yeasts in the form of “pied de cuve” [21,22]. The “pied de cuve” technique involves harvesting grapes several days prior to the main harvest and fermenting the must in a small volume to propagate the autochthonous microbiota, which will then be used to inoculate the main fermentation vessel. This approach helps preserve the microbial diversity associated with the terroir while allowing for a certain degree of microbial control [19]. Alvarez-Barragan et al. [21] conducted the spontaneous fermentation, “pied de cuve” and controlled fermentation (using commercial starter yeast cultures) of musts to produce corresponding Chardonnay and Pinot Noir wines. These wines were subsequently compared at the metabolomic, chemical, and sensory levels. The results revealed that the choice of yeast significantly influenced the wines’ molecular composition with distinct chemical profiles for each fermentation method. Sensory analysis of Chardonnay wines showed higher fruit intensity in wines obtained by controlled and spontaneous fermentation compared to those fermented using the “pied de cuve” fermentation method. These findings challenge the common belief among winemakers that the use of autochthonous yeast will result in wines of higher complexity and distinctiveness than the use of commercial starter yeast cultures.

3. Development of Wine Yeast Starter Cultures for Optimizing Controlled Fermentation

Prior to the availability of commercial wine yeast starter cultures (i.e., active dry yeasts), winemakers were required to propagate starter cultures from their own microbiological collections within the wine cellars to perform inoculated and controlled fermentation. This process involved inoculating pure yeast cultures into pasteurized juice and propagating the yeast in several stages over several weeks. Once the appropriate quantity of starter culture was prepared, it was tested for microbial contamination before being used to inoculate the must. The main challenge with this propagation method was maintaining the purity of the yeast culture, as contamination risk was high. Additionally, the process required considerable time, expertise, resources and labor [23].

In view of the difficulties mentioned above, the development of commercially available yeasts was a significant step forward. This was preceded by research that indicated the possibility of using pressed yeasts, similar to those used in the baking industry. The problem, however, was their short shelf life due to their high moisture content (70%). Unlike the baking industry, which uses yeast all year round, wineries require large quantities of yeast in a relatively short period of time. This problem was solved in 1963, when the drying of wine yeasts was successfully introduced [23]. This first generation of dry wine yeasts also comprised strains recommended for restarting fermentations that had stalled (i.e., stuck fermentation). Although the first commercial wine yeasts were marketed worldwide as all-purpose yeasts, their application was only partially successful. Given the characteristics expected of modern wine yeasts, some of the first-generation yeasts would probably be rejected in selection today [24].

As it became evident that these strains were not universally effective for must fermentation, the need arose to develop second-generation strains that were more adapted to the specific wine regions and their grape varieties. Consequently, in the mid-1970s, new strains of dry wine yeasts were developed, with technological and oenological characteristics that had a predictable impact on wine quality. Each of these strains exhibited desirable traits, the effects of which on wine were well studied, including the ability to ferment at low temperatures, produce low foam and good settling, display a killer factor, produce aroma compounds, and demonstrate efficient flocculation [25].

The third generation of wine yeasts, which has been produced more recently, meets modern requirements and is characterized by specific enzymatic activities. Thus, it contributes to a stronger release of the aromatic substances originating from the grapes (e.g., the yeast enzyme β-glycosidase can release bound terpenes from the Muscat and Sauvignon varieties) [25].

By the end of the 20th century, the inoculation of individual yeast starter cultures (mainly of the species S. cerevisiae) had become a widespread practice, and dozens of different yeast strains were on the market [26].

A historical overview of the development of wine yeast starter cultures is presented in Figure 1.

There is already quite an extensive literature on the use of commercial yeast starter cultures in the production of wine from grapes [2,29] and the research on the isolation and characterization of new wine yeast strains has also been carried out in Croatia [30,31,32,33,34,35]. Research on the impact of specific yeast strains on the properties of fruit wines has been documented in the scientific literature [36,37,38,39,40,41,42,43]. However, the body of literature remains significantly smaller than that on grape wines, with a limited number of studies comparing different starter cultures in the production of specific types of fruit wines, highlighting a potentially valuable research area.

The scientific literature on Croatian blackberry wines is limited, with publications emerging only in the last 15 years [9,10,41,44,45,46,47,48,49,50,51]. Although the type of yeast and the conditions prevailing during fermentation, along with the type of fruit used, significantly influence the aroma and taste of fruit wine, only two published studies, to our knowledge, have examined various commercially available wine yeasts for the production of Croatian blackberry wines [41,50]. Given the considerable differences in the composition of various fruit types (e.g., cultivated and wild blackberry [52]), the yeast strains used for fruit wine production must adapt to diverse environmental conditions, such as variations in sugar type and concentration, the presence of organic acids, and other factors.

All three previously described generations of selected wine yeasts (starter cultures), primarily consist of yeasts belonging to the Saccharomyces genus, predominantly Saccharomyces cerevisiae strains, due to their higher resilience to stresses [53]. Saccharomyces cerevisiae is also the most extensively studied yeast in fruit wine production [54].

About a quarter of a century ago, some authors pointed out the possible oenological potential of using alternative yeasts, including Saccharomyces cerevisiae strains isolated from non-oenological environments, as well as non-Saccharomyces cerevisiae strains, highlighting interesting and novel oenological properties that could have a positive influence on the sensory profile of wines [13].

Yeasts other than Saccharomyces (i.e., non-Saccharomyces yeasts) have traditionally been considered undesirable in winemaking due to the production of many undesirable metabolites with negative aroma characteristics. However, recent studies have demonstrated that certain non-Saccharomyces yeasts can exhibit intriguing aroma profiles and enzymatic activities that positively influence wine composition. As a result, a fourth generation of wine yeasts has emerged, with some non-Saccharomyces yeasts being selected and commercially produced over the past 10–15 years (

Table 1. Most commonly used non-Saccharomyces yeasts for wine and fruit wines.

Non-Saccharomyces Yeast	Wine Type	References
Hanseniaspora osmophila Lachancea thermotolerans Metschnikowia pulcherrima Pichia fermentans Starmerella bacillaris Torulaspora delbrueckii Zygotorulaspora florentina	Red wine	[59] [59] [59,60] [59] [59] [59,60]
Candida zemplinina	White wine	[61]
Candida californica		[61]
Hanseniaspora meyeri		[62]
Hanseniaspora osmophila		[61,62]
Hanseniaspora uvarum		[61]
Pichia guilliermondii		[62]
Pichia kudriavzevii		[62]
Schizosaccharomyces pombe		[63]
Torulaspora delbrueckii		[62,64]
Wickerhamomyces anomalus		[62]
Candida ethanolica	Fruit wine	[65]
Candida glabrata		[66]
Cyberlindnera fabianii		[67]
Hanseniaspora guilliermondii		[65]
Hanseniaspora occidentalis		[68]
Hanseniaspora opuntiae		[68]
Hanseniaspora uvarum		[68]
Hanseniaspora thailandica		[65]
Kloeckera apiculata		[69]
Metschnikowia fructicola		[70]
Metschnikowia pulcherrima		[69,70,71]
Meyerozyma guilliermondii		[72]
Pichia fermentans		[73]
Pichia kudriavzevii		[68,72]
Torulaspora delbrueckii		[67,70,71]
Wickerhamomyces anomalus		[67]
Zygosaccharomyces rouxii		[72]

). They can influence the color and aroma, or contribute to the quality and stability of the wine [55,56,57,58].

There are two methods of inoculation when using non-Saccharomyces yeasts in mixed starters: (1) co-inoculation (mixed fermentation), i.e., inoculation of selected non-Saccharomyces yeasts at a high cell concentration simultaneously with S. cerevisiae yeast; and (2) sequential inoculation, where non-Saccharomyces yeasts (one or more selected strains) are inoculated at high levels in the first step, and allowed to ferment for a certain time, while in the second step, S. cerevisiae is inoculated to take control of and complete the fermentation [17]. Both approaches are feasible; however, potential interactions between yeasts must be considered when selecting the most appropriate inoculation strategy. Despite previous research on wine aroma, results for mixed cultures consisting of multiple non-Saccharomyces species in combination with S. cerevisiae, mimicking the complex yeast microbiota in fermenting must, are sometimes inconsistent [28].

Non-Saccharomyces yeasts, such as Pichia kluyveri, Pichia anomala, Torulospora delbrueckii, Metchnikowia pulcherrima, Lachancea thermotolerans, Candida stellata, Hanseniaspora/Kloeckera spp., Wickerhamomyces anomalus and Schizosaccharomyces pombe, are among the species used for mixed or sequential fermentation with S. cerevisiae [74]. Although non-Saccharomyces yeasts can be responsible for incomplete fermentation (higher residual sugar levels) and higher acetic acid and ethyl acetate levels [75], they can produce different volatiles that better coordinate aromas and improve the quality of the wine with more terroir and distinctive characteristics [43].

Research on non-Saccharomyces yeasts for grape wine production has also been initiated in Croatia. This includes the investigation of the fermentative potential of autochthonous non-Saccharomyces yeasts, previously isolated from Maraština grapes, for their possible contribution to the organoleptic properties of wine, with the aim of selecting a potential starter culture [76,77,78].

The literature on berry and other fruit wines produced with non-Saccharomyces yeasts remains significantly more limited compared to that on grape wines [65,66,68,69,71,79]. In blueberry wine, Che et al. [66] concluded that the co-fermentation of S. cerevisiae with C. glabrata E4 and Pichia anomala E2 enhanced wine quality and flavor (sweetness, fruitiness, and florality) by increasing the content of isoamylol, 2-phenylethanol, isoamyl acetate, ethyl acetate, and ethyl laurate. Additionally, Wickerhamomyces anomalus was found to increase the total phenol and total flavonoid content. In orange wines, Hanseniaspora strains contributed to aroma development through the production of ethyl caprylate, ethyl hexanoate, and isoamyl acetate [79]. Recently, studies have also been conducted on Chunjian citrus fruits [69], orange wine [68], and tangerine wine [65].

The development of identification methods has confirmed the existence of Saccharomyces and non-Saccharomyces yeast strains that are characteristic of specific oenological regions, supporting the hypothesis that these strains may be better adapted to the particular conditions of winemaking. In countries with a long tradition of winemaking, locally selected, autochthonous yeast strains are used, which, in addition to desirable technological properties, also possess a metabolic profile suited to the production of specific wines. Yeast strains characteristic of certain oenological regions can be significantly more competitive than other strains, as they are better adapted to the winemaking conditions of the wine region from which they were isolated [80,81]. The isolation and identification of local yeast strains to develop region-specific wine starter cultures remains the preferred method for achieving the uniqueness of locally produced wines [82].

Currently, hundreds of selected yeast strains (Saccharomyces and non-Saccharomyces) are commercially available worldwide and are produced by several international companies. These strains are isolated from wineries or research centers, with many of unknown origin. Due to the wide availability of commercial strains for grape wines, the selection of an appropriate starter culture can be challenging. As a result, most starter yeast culture producers provide guides or tools on their respective websites to assist winemakers in selecting commercial yeast strains for wine production. Based on the desired wine type and sensory profile, suitable yeast strains are suggested by manufacturers to optimize the production process. On the other hand, commercial yeast starter cultures exclusively selected and developed for fruit wines, except cider, are largely absent from the market, which is why fruit wine producers resort to using starter cultures intended for grape wines.

Table 2. An overview of the differences in the number of starter cultures available for grape and fruit winemaking, based on the example of four starter culture producers.

Producer	Grape Wine Yeast Starter Cultures	Fruit Wine Yeast Starter Cultures
LAFFORT® [83]	Total: 28 Saccharomyces strains 4 non-Saccharomyces strains (ZYMAFLORETM, ACTIFLORETM) White wines: 19 Saccharomyces strains 3 non-Saccharomyces strains Rose wines: 19 Saccharomyces strains 3 non-Saccharomyces strains Red wines: 16 Saccharomyces strains 2 non-Saccharomyces strains Sparkling wines: 13 Saccharomyces strains	No yeast strains exclusively selected for fruit wines (ZYMAFLORETM, ACTIFLORETM) Cider: 8 Saccharomyces strain 1 non-Saccharomyces strain Other fruit wines: 9 Saccharomyces strains 1 non-Saccharomyces strain
LALLEMAND OENOLOGY [84]	Total: 45 Saccharomyces strains 4 non-Saccharomyces strains (LalvinTM, UvafermTM, Level2TM, EnofermTM, IONYS wfTM, etc.) White wines: 21 Saccharomyces strains 3 non-Saccharomyces strains Rose wines: 13 Saccharomyces strains 2 non-Saccharomyces strains Red wines: 23 Saccharomyces strains 2 non-Saccharomyces strains	No yeast strains exclusively selected for fruit wines (LalvinTM, UvafermTM, Level2TM, EnofermTM, IONYS wfTM, etc.) Cider: 8 Saccharomyces strains 1 non-Saccharomyces strain Other fruit wines: No strains are specifically recommended for a particular type of fruit wine
FERMENTIS by Lesaffre [85]	Total: 19 Saccharomyces strains (SafŒnoTM) White wines: 9 Saccharomyces strains Rose wines: 5 Saccharomyces strains Red wines: 8 Saccharomyces strains Sparkling wines: 3 Saccharomyces strains	Except for cider, no yeast strains exclusively selected for fruit wines Cider: Total: 5 Saccharomyces strains (SafCiderTM) Other fruit wines: No strains are specifically recommended for a particular type of fruit wine
AEB [86]	Total: 30 Saccharomyces strains (Fermol®, Zymasil®) 6 non-Saccharomyces strains (Levulia®, NS Ferm®, Primaflora®) White wines: 23 Saccharomyces strains Rose/light red wines: 13 Saccharomyces strains Big Red wines: 9 Saccharomyces strains Sparkling wines: 4 Saccharomyces strains	Except for cider, no yeast strains exclusively selected for fruit wines Cider: Total: 3 Saccharomyces strains (Zymasil Special Cider, NXT Zymasil Cider NXT Zymasil Cider Bayanus) Other fruit wines: No strains are specifically recommended for a particular type of fruit wine

provides an overview of the differences in the number of starter cultures available for grape and fruit winemaking, based on the example of four major yeast starter culture producers. It can be seen from the table that, among the yeasts selected for grape wine production, some are specifically declared by manufacturers as suitable for certain fruit wines. However, no examples are found of yeasts exclusively selected for fruit wines, except for those designated for cider making.

4. The Forms of Commercial Wine Yeast Starter Cultures

Most yeast starter cultures are pure, meaning they contain only a single yeast strain, primarily strains of Saccharomyces cerevisiae [87,88]. Pure starter cultures are generally used in two forms: active dry yeast and liquid yeast [25,89]. Additionally, other forms of commercial yeast cultures available for distribution have been reported in the literature, including yeast grown on solid or liquid media, lyophilized yeast, concentrated yeast paste, and immobilized yeast [25].

Liquid wine yeast starter cultures, which are not subjected to drying, consist only of yeasts that are not suitable for drying or that have other properties suitable for winemaking (e.g., more active and, thus, more competitive against other microflora, convenient for fermentation of dry berry selections, ice wines or sparkling wines). These yeast starters are produced in the form of liquid cultures by the winery, or commercially supplied by local wine laboratories or institutes. Production begins with the transfer of a pure culture from solid to liquid media, followed by cultivation in small batches, e.g., 1–10 L. The advantage of yeast cultivated in liquid media is that the preparation time in the winery is shortened, the fermenting culture is active, it contains a high population of viable cells and is, therefore, more competitive against contaminating microorganisms. The downside is that the liquid culture is unsuitable for transportation—it cannot be sealed as it ferments and releases CO₂. Due to its limited viability, the good production properties could soon be lost if the liquid culture is not used immediately while the yeasts are in the exponential growth phase. Therefore, liquid wine yeast cultures can only be used for a short period of time and their market is limited due to their short shelf life [87,89]. The paste prepared in the form of a concentrated paste, similar to that used for baking, differs from the liquid culture as it is cultivated under aerobic conditions. The paste contains only around 30% of the dry weight and is, therefore, more unstable than dry yeast in powder form. Therefore, it cannot be marketed on a large scale in the wine industry [25].

Wine yeasts suitable for drying are cultivated industrially in a medium containing molasses under aerobic conditions, separated from the medium, washed and dried. After drying in the form of granules with a moisture content of 6–8% (w/w), commercial wine yeasts are packaged and sealed under vacuum or nitrogen. Such yeasts do not need to be propagated but are rehydrated or added directly to the must or pomace (marc). The amount of added granules is indicated on the packaging, and in certain cases, larger quantities are required, e.g., if the fermentation temperature is below 12 °C, if undesirable microorganisms are present or if fermentation is inhibited or stalled [90,91].

When dry yeasts were first used in winemaking, they were mainly selected for their technological advantages. Today, their sensory properties and the contribution of the yeast’s metabolic profile to the overall quality of the wine are equally important. The use of selected yeast strains influences the overall quality of the wine, i.e., they produce metabolites that influence the composition and the sensory properties of wine [92,93], such as esters, higher alcohols, carbonyl compounds, volatile acids, volatile phenolic compounds and sulfur compounds [88]. Considering the possible differences in the biosynthesis of volatile compounds between different yeast species and strains, it is important to select the best strain whose metabolic activities during fermentation result in high-quality wine [3]. Although there is a high probability that the inoculated S. cerevisiae yeast will prevail during fermentation, inoculation does not necessarily guarantee 100% dominance of the strain or its exclusive contribution to fermentation. This is significantly influenced by the population of autochthonous yeasts present in the juice, as well as the adaptation of the commercial yeast strain to the environment [27].

The biotechnological principles of commercial wine yeast cultivation and drying, which ensure the subsequent fermentation characteristics and wine quality, are described in more detail in the literature and are not the subject of this paper [22,87,94].

The production of active dry yeast starters has been optimized for S cerevisiae strains, which can lead to problems in the propagation of non-Saccharomyces yeasts. Therefore, for the industrial cultivation of non-Saccharomyces wine yeasts, modifications should be made to the cultivation media to ensure good propagation of the yeast biomass and avoid adverse effects on the quality of the wine [95]. In addition, it is necessary to optimize the drying process of non-Saccharomyces yeasts to allow their optimal activity and prevent the deterioration of wine quality [96].

Yeast grown on solid medium is the simplest method for maintaining and distributing pure cultures from a supplier, typically on a slant or Petri plate; however, further propagation is required before fermentation in the winery.

Lyophilized yeast is prepared from a pure yeast culture that is cultivated in large quantities on a sugar-rich medium, harvested by centrifugation, resuspended in an appropriate protective solution, and rapidly frozen at −40 °C at a rate of 3 °C per minute. The ice is then sublimated and removed by slowly heating the frozen cell mass under a vacuum. The viability of the lyophilized culture ranges from 5 to 10 years, with the survival rate depending on the yeast strain, protective solution, and storage temperature. After rehydration, lyophilized yeast can be used directly or cultured according to the manufacturer’s instructions [25].

The use of immobilized yeasts in winemaking may offer some advantages over the use of the free yeast cell method. The most extensively studied methods for yeast immobilization include the use of natural carriers (e.g., lignocellulosic materials), organic carriers (e.g., alginate), inorganic carriers (e.g., porous ceramics), membrane-based systems and multifunctional agents. However, the current industrial application of immobilized yeast is still rare [97].

5. Advantages and Disadvantages of Using Commercial Wine Yeasts in Controlled (Inoculated) Fermentation

As already stated, the use of starter cultures of wine yeasts (large number of cells of the selected yeast) suppresses the growth of wild yeasts present on the fruit and in the must, while the added yeast strain grows rapidly and multiplies in greater numbers. Therefore, the starter culture used determines the characteristics of the final product, the wines have a purer color and the varietal characteristics are more pronounced [98].

If fermentation stalls, inoculation with a yeast starter culture is required. Starter cultures must also be added if the grapes/fruit are heavily infested with molds, if yeast-toxic fungicides have been used or if must with a high sugar content is used for fermentation. It is also necessary after thermovinification (applying high temperatures to crushed grapes before the start of the fermentation) and during the fermentation of pasteurized must or diluted concentrate. In the production of sparkling wine, inoculation with starter cultures is necessary to enable secondary fermentation [89].

The advantages of using commercial wine yeasts are as follows [23,98]:

• Fermentation starts faster because the inoculated strain quickly becomes dominant.
• The time of fermentation start and duration, as well as the maximum fermentation rate, can be predicted so that problems during fermentation can be detected more quickly and appropriate measures can be taken.
• The negative effects of naturally occurring microflora on the organoleptic properties of the wine are reduced.
• If the aroma of the grapes is to be emphasized, then so-called “neutral” yeasts can be used.
• If a particular aroma is to be achieved, yeasts that produce larger quantities of aroma compounds can be used.

However, there are some shortcomings, mentioned in the literature, of using commercial wine yeasts [87,91,98]:

• The use of the same yeasts for the fermentation of different types of must can lead to an already mentioned “uniformity” of the wine, i.e., similar characteristics, which is undesirable for some categories of consumers.
• The fermentation rate is too high if too much yeast is inoculated, leading to heating and loss of volatile aroma compounds.
• The possibility of an overly pronounced yeast aroma if too much yeast is inoculated.

Despite the advantages of commercial yeast cultures, winemakers are still divided on the philosophy and practice of using starter cultures [23]. On the one hand, some winemakers rely solely on autochthonous yeasts and bacteria to obtain a unique product. Others prefer to stimulate the growth of yeasts from other genera (i.e., non-Saccharomyces yeasts) at the beginning of fermentation and later inoculate with yeasts from the Saccharomyces genus. Some of the wine producers use Saccharomyces starter cultures, although in smaller quantities than recommended, while the vast majority of winemakers use active yeast prepared according to the manufacturer’s recommendations. This is in line with the prevailing opinion that the use of dry wine yeast ensures a faster start of fermentation while promoting the production of wines with consistent quality [27].

6. Criteria for the Selection of Wine/Fruit Wine Yeasts

As already mentioned, the commercial yeast strains selected for wine production mainly belong to the species Saccharomyces cerevisiae. Most of these strains were isolated from fermenting musts or pomace, and each of them was tested for suitability and cultivated to obtain the desirable characteristics [98]. The selection of yeasts is often preceded by their identification, which is primarily performed by molecular methods [99]. Wang et al. [100] correlated microbial community and aroma compounds by monitoring dynamic changes of the microbial community structure in must samples inoculated with four different starters. Identification of yeast starter strains was performed by high-quality DNA, PCR amplification, and HTS. Correlations between microbiota and produced volatiles (determined by HS-SPME combined with GC-MS) were performed by the PLS-DA model.

The production of quality fruit wines requires suitable modern analytical methods and sensory analysis. There are a large number of possible analyses for the evaluation of wines and fruit wines. To ensure and maintain the identity and quality standards of the wine, various analyses are performed during production and on the wine itself: physical (e.g., pH, density), chemical (e.g., ethanol, acids, carbohydrates), microbiological (e.g., presence of desirable yeasts and bacteria, presence of contaminating microorganisms), or sensory (product acceptability). Dias et al. [101] published an overview of methodologies for fruit wine analysis, which include physicochemical, chromatographic, microbiological (classical and molecular techniques) and sensory analysis. A variety of modern analytical methods have been published by Pozo-Bayón and Muñoz González [102].

The most popular methods for yeast identification are molecular (RFLP of ribosomal ITS region or mitochondrial DNA and others) [103,104,105] and MALDI TOF mass spectrometry [106,107], while classical selection methods remain useful for excluding unsuitable strains from the selection process.

Simple tests are carried out to determine which yeasts have the best potential for wine fermentation. The exclusion criteria are high acetic acid and H₂S production, low tolerance to ethanol and SO₂, foam formation, and no or low enzyme activity. After the initial selection phase, the best strains are identified and tested in must fermentation [82,99,100,102,108]. In addition to the characterization of Saccharomyces strains, the oenological potential of certain non-Saccharomyces strains (e.g., Hanseniaspora, Candida, Pichia, Zygosaccharomyces, Kluyveromyces) is increasingly being investigated [109,110,111]. Although ethanol content is important in winemaking, some non-Saccharomyces yeasts (M. pulcherrima Mp51 and MpFA) have reduced ethanol yields by 1.17 to 1.39%, respectively, when sequentially inoculated with S. cerevisiae [112]. This can be used as a selection criterion for wines with reduced ethanol content to address the health concerns of some consumers.

As already mentioned, better control of fermentation through the use of selected yeast strains is one of the prerequisites for the production of high-quality wines [16,18,41]. The oenological properties of wine yeasts can be divided into two groups, namely technological and qualitative. Both groups of properties must be taken into account when selecting wine yeast. The technological properties influence the efficiency of fermentation, while the qualitative properties determine the chemical composition and sensory characteristics of the wine. Determining these properties is very important because yeasts differ from each other in their characteristics. The technological properties can be determined by monitoring the progress of fermentation, while the qualitative properties are assessed by the chemical composition of the wine. Properties that have recently been evaluated include the metabolic properties of yeast that impact human health, such as the potential for ethyl carbamate and urea production, sulfite production and biogenic amines production (e.g., histamine) [113,114].

It is of the utmost importance to minimize the production of undesirable compounds with negative impacts on humans [108]. Examples of such compounds are ethyl carbamate and biogenic amines. Ethyl carbamate (EC, also known as urethane) is a probable human carcinogen naturally occurring in wine, and reducing its levels is important from a food safety perspective. In alcoholic beverages, EC is formed during the fermentation and storage phase when ethanol reacts with urea, citrulline and carbamyl phosphate, which are formed by the arginine metabolism of wine yeasts and lactic acid bacteria [108,115]. However, the main pathway of EC formation in wine is the reaction of ethanol with urea, which is formed during yeast catabolism of arginine. The addition of urea as a nitrogen supplement to grape must is prohibited in most wine-producing countries. As the main precursor of EC, urea can be degraded enzymatically, and the addition of commercial preparations of the enzyme acid urease is permitted by the International Organisation of Vine and Wine and EEC legislation [108]. Moreover, the selection of wine yeasts with low urea production or even urea degradation could be a useful strategy to limit EC concentrations.

Biogenic amines (BAs) are known to be a possible cause of headaches, heart palpitations, vomiting, diarrhea, and hypertensive crises. Their toxicity depends on the individual, as well as on the specific biogenic amine and its concentration [108]. It is generally believed that BAs are mainly derived from malolactic fermentation or spoilage bacteria and that malolactic bacteria are selected for BA formation. Yeasts can also contribute significantly to the BA content in wine and the amount of each BA produced is strain-dependent [108,116]. Therefore, one of the approaches currently being explored to reduce BAs levels is the selection of an appropriate starter culture.

Schizosaccharomyces pombe is an urease-positive yeast that plays a beneficial role in reducing the urea content of EC. Additionally, certain S. pombe strains can degrade malic acid almost completely. These yeasts may be advantageous in preventing the synthesis of both BA and EC [116]. However, some of the strains also exhibit certain limitations, including reduced fermentation kinetics and the potential biosynthesis of metabolites associated with off-flavors [117], such as H₂S, acetic acid, acetaldehyde, acetoin and ethyl acetate [63], which is probably why the commercial strains of this species are rarely found on the market. Among 75 S. pombe strains tested, Benito et al. [118] selected three very promising strains, with potentially useful winemaking properties. In addition to the appropriate volatile aroma profiles, the three S. pombe strains are beneficial with respect to food safety regarding low BA and EC production. In the mentioned study, S. pombe yeasts did not produce higher levels of BA than S. cerevisiae. The malic dehydrogenase activity, which is a general advantageous characteristic found in almost all S. pombe strains, allows malic acid deacidification by converting malic acid to ethanol and CO₂ without the need for later malolactic fermentation, which could bring increased BA levels. In addition, urease activity of S. pombe plays a beneficial role in reducing the content of urea as a precursor of EC. At the same time, the fermentations performed by S. pombe resulted in very similar ethanol levels to those performed by the examined S. cerevisiae strains.

In the modern selection of yeast strains, those with potential harmful effects on human health should be excluded, while strains that positively influence the health-promoting and aromatic properties of wine should be prioritized. During wine fermentation, the total polyphenol content is reduced by adsorption to yeast cell wall polymers and subsequent separation. Four S. cerevisiae strains (of 29 analyzed) showed a significantly (p < 0.05) increased amount of anthocyanins, quercetin and trans-coutaric acid, minimal volatile acidity (<0.2 g/L), absence of undesirable metabolites and a balanced volatile profile, resulting in a wine with enriched polyphenolic content [119].

The final stage of yeast characterization is the sensory analysis of the wine produced. The sensory characteristics of wines, such as taste, astringency, bitterness, and color, are significantly influenced by various phenolic compounds present in fruits and grapes. Phenolic compounds in both grape and fruit wines are considered key bioactive constituents with significant potential for health benefits and are most commonly associated with the positive effects of moderate wine consumption [120,121]. Since the production of fruit wines is almost identical to that of grape wines, significant changes occur in the composition of the raw material (fruit juice/must) during the vinification process [93]. Both fermentation and maturation lead to a transformation of the original compounds into secondary metabolites, which can affect the quality of the final product [45,116,122]. The choice of fruit also affects the composition of the fruit wine, as not all fruit varieties are suitable for fermentation, i.e., the production of fruit wines where the lower sugar concentration and pH value are the limiting factors [123]. Therefore, a variety of factors affecting the sensory and phytochemical properties of fruit must be considered in the production of fruit wines. It should be noted that changes in the composition and antioxidant properties of foods can affect consumer choice and acceptance.

The presence of organic acids influences the organoleptic properties of musts and fruit wines. Similarly, phenolic compounds are not the only constituents of fruit wines; however, they contribute significantly to the sensory characteristics and the color and flavor of fruit wines. Moreover, different applied technologies and functional properties can influence the specific “phenolic pattern” of the wine [124,125,126].

Nowadays, the role of selected yeasts goes beyond the mere fermentation of sugar to ethanol and various requirements must be met [119,127]. During alcoholic fermentation of must, the ethanol concentration gradually increases, and pH, temperature and pressure have potentially unfavorable effects on yeast. In addition, competition between commercial strains and autochthonous microorganisms and nutrient limitation may occur during the fermentation process. To ensure a smooth process and to obtain wine of the desired quality, the yeast must be provided with sufficient nutrients for growth and fermentation. In addition to the fermentation conditions set by the winemaker, the producer of commercial wine yeast is responsible for ensuring cultivation and drying conditions that guarantee good yeast activity during fermentation [128].

The most important property of a yeast strain is its fermentation ability. Sometimes, the yeast must be capable of fermenting must with more than 200 g/L of sugar under normal winemaking conditions so that the concentration of unfermented sugar decreases to 0.2 to 2 g/L. Yeasts can also be selected for a variety of other characteristics, some of which are listed in

Table 3. Criteria for the selection of yeast strains for commercial use [2,3,94,129].

	Desirable Criteria	Undesirable Criteria
Qualitative properties	▪ production of desirable fruit aromas and esters ▪ production of β–glucosidase ▪ production of glycerol ▪ production of mannoproteins	▪ production of SO2 ▪ production of H2S ▪ production of sulfur compounds ▪ production of volatile acids and ethyl acetate ▪ production of compounds that bind SO2 (acetaldehyde, pyruvate…) ▪ formation of ethyl carbamate precursors ▪ production of polyphenol oxidase ▪ production of biogenic amines
For special purposes	▪ degradation of malic acid ▪ formation of lactic acid ▪ formation of isoamyl acetate ▪ rapid autolysis	-
Technological properties	▪ complete fermentation of sugar ▪ high tolerance to ethanol ▪ resistance to SO2 ▪ minimal lag phase after rehydration ▪ fermentation at low temperatures ▪ tolerance to high temperatures ▪ activity during fermentation ▪ presence of killer factors	▪ foaming ▪ biofilm formation ▪ activity during fermentation
For special purposes	▪ agglomerations properties ▪ sedimentation properties	-

.

Some of the investigated yeast properties are always desirable, while others are always undesirable in wine production. Researching all these properties in detail is very time-consuming and not feasible in practice. The characterization of yeast strains requires sophisticated equipment and trained personnel. Since certain types of wine require specific combinations of wine yeast characteristics, research focuses only on some of the numerous characteristics [130]. When selecting wine yeast strains, the production technology and the type of target product must be taken into account, i.e., whether a particular selected yeast strain is to be used for the production of grape wine, sparkling wine, botrytized wines with high sugar content, or a fruit wine [82].

The volatile compounds in wine are responsible for the wine’s aroma. The analysis of these compounds requires the use of different analytical techniques, depending on the type of compound and its concentration [131]. In addition to ethanol, a number of by-products are also formed during alcoholic fermentation. These include carbonyl compounds, alcohols, esters, acids and acetals, all of which affect the quality of the end product (wine). Aroma compounds make up only a small part of the overall chemical composition of fruit, although they have a decisive influence on the sensory properties of fruit and fruit products (e.g., fruit wine). Many aroma compounds originally present in the fruit are transformed during the fruit processing (fermentation, wine aging), and the final aroma is determined by the combined effect of the compounds originally present and those resulting from the transformation. The chemical profile of wine originates from grapes/fruit, the fermentation microflora (especially yeast S. cerevisiae), malolactic fermentation and aging (maturing), as well as the storage conditions of the wine. Much of the literature deals with the aroma and taste of grape wines [132,133], while the data on the aroma and taste of fruit wines are limited, especially blackberry wines of the Croatian region [47]. The literature gap on the use of non-Saccharomyces yeasts in blackberry winemaking can be addressed based on the research results of Mančić et al., who isolated yeasts from wild and cultivated blackberries, but also evaluated the fermentative potential of autochthonous Candida famata isolates in grape must, and not in blackberry wine production [134].

The metabolic pathways for the formation of various aroma compounds in yeast are described in the literature [91,135]. Generally, the compounds produced by the selected yeast strain should improve the quality of the wine, and undoubtedly not reduce it. The production of glycerol is desirable, while the production of acetic acid, ethyl acetate and higher alcohols should be insignificant, as well as the formation of H₂S, SO₂ and compounds that bind SO₂. The yeast should not cause excessive foaming and should settle quickly after fermentation [98].

Due to global warming, the production of organic acids is gaining importance as one of the selection criteria for wine yeasts. Global warming is associated with a lower content of organic acids in grapes/fruit and wine, which also leads to a higher pH of the wine. It changes the free and molecular sulfur dioxide, the color, and the sensory aspects of the wine. The most commonly used acidification method is chemical acidification, followed by physical acidification and microbiological acidification; however, the microbiological method can also be used to solve the problem of low wine acidity. The first option is the use of S. cerevisiae strains that can produce small amounts of organic acids (malic, lactic or succinic acid). The second option is to use Lachancea thermotolerans (non-Saccharomyces yeast, formerly known as Kluyveromyces thermotolerans), which can increase the lactic acid concentration and lower the pH. Some other non-Saccharomyces yeasts have been shown to be useful for the acidification of wine, e.g., Candida zemplinina, formerly known as Starmerella bacillaris, applied in co-culture or sequential fermentation with S. cerevisiae. Positive results have also been obtained with Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia fructicola in sequential fermentations [136].

7. Future Prospects

A potential direction for future research on wine yeast starter cultures could involve further exploration of the potential of non-Saccharomyces yeasts and their combination with Saccharomyces yeasts in winemaking, as well as the isolation and selection of new autochthonous yeast strains specific to certain terroirs, with superior traits tailored to meet consumer preferences. In the context of fruit wines, future research might focus on the isolation, selection, and subsequent use of both Saccharomyces and non-Saccharomyces yeast strains, specifically customized for particular fruit varieties to optimize fermentation and enhance the distinct characteristics of fruit wines, offering consumers a broader range of flavors. One of the topics for both grape and fruit wines will likely include the exploration of how different yeast strains perform when raw materials are influenced by changing climatic conditions while ensuring the continued production of high-quality wines in the face of climate change challenges.

8. Conclusions

Advances in winemaking technology and a deeper understanding of yeast physiology have significantly improved the quality of grape and fruit wines. Although fruit wines have a smaller market share and are less recognized, their importance is increasing due to the health benefits of fruits. Although some winemakers still prefer spontaneous fermentation or the use of other techniques that include the application of autochthonous yeast, the use of selected commercial wine yeast starter cultures predominates in modern winemaking ensuring the reliable fermentation kinetics and reproducibility of the fermentation process that yields the final high-quality product. The selection criteria for wine yeasts have changed over time in accordance with the increasing growing demands of consumers. Modern criteria not only include desirable technological characteristics of the yeast and the achievement of certain secondary flavors but also consider the impact of the wine’s composition on consumer health. These evolving requirements present new challenges for both grape and fruit wine producers as well as producers of wine yeast starter cultures.