Role of Yeasts in the Brewing Process: Tradition and Innovation

: Nowadays, in the beer sector, there is a wide range of products, which differ for the technologies adopted, raw materials used, and microorganisms involved in the fermentation processes. The quality of beer is directly related to the fermentation activity of yeasts that, in addition to the production of alcohol, synthesize various compounds that contribute to the deﬁnition of the compositional and organoleptic characteristics. The microbrewing phenomenon (craft revolution) and the growing demand for innovative and specialty beers has stimulated researchers and brewers to select new yeast strains possessing particular technological and metabolic characteristics. Up until a few years ago, the selection of starter yeasts used in brewing was exclusively carried out on strains belonging to the genus Saccharomyces . However, some non- Saccharomyces yeasts have a speciﬁc enzymatic activity that can help to typify the taste and beer aroma. These yeasts, used as a single or mixed starter with Saccharomyces strains, represent a new biotechnological resource to produce beers with particular properties. This review describes the role of Saccharomyces and non- Saccharomyces yeasts in brewing, and some future biotechnological perspectives.


Introduction
Beer is a fermented alcoholic beverage which has been produced for thousands of years, and it is consumed worldwide. Beer is obtained by the fermentation of yeasts of a must consisting of water, malted cereals (usually barley and wheat), and hops. The brewing process basically includes the following stages: malting (in which cereal is converted into malt), mashing (during this phase the malt enzymes convert the grain starches into fermentable sugars), alcoholic fermentation and maturation.
The main stages in the brewing process are: wort production, alcoholic fermentation and maturation, processing, and stabilization of the beer.
The wort transforms into beer during alcoholic fermentation and maturation, which are the longest processes in brewing. The primary fermentation lasts between 3 and 6 days, and the maturation up to 2 weeks depending on the fermentation type and the equipment used. Ethanol fermentation occurs as a result of enzymatic activity of the yeast at the Embden-Meyerhof-Parnas pathway, which leads to glucose conversion to pyruvate. Under anaerobic conditions, the yeasts convert pyruvate to ethanol and CO 2 . In aerobic conditions, yeasts consume sugars, mainly for biomass accumulation and CO 2 production.
The yeasts uptake the carbohydrates of wort in a specific sequence: monosaccharides (glucose and fructose), disaccharides (sucrose and maltose), and trisaccharide maltotriose, and ferment them in the same order. A very small amount of maltotriose is used for the formation of reserve polysaccharides (glycogen and trehalose). The amino acids assimilated by yeast are used for the synthesis of proteins, enzymes, and new cells. The fermentation byproducts: carbonyl compounds, higher alcohols, esters, organic acids, and sulfur-containing compounds determine the flavor profile and affect the quality of the beer.  The Saccharomyces yeasts, involved in alcoholic fermentation processes, play a decisive role in the organoleptic characterization of the products [2].
For large-scale beverage fermentation, as in brewing, winemaking, and distilled spirit production, pure cultures of selected strains of Saccharomyces spp. are usually used, whereas in smaller-scale (artisanal) processes, spontaneous fermentation is often developed by indigenous microflora found in the raw material [3].
The Saccharomyces genus contains some important species for the food industry, namely, Saccharomyces cerevisiae (used in winemaking and ale beer brewing), Saccharomyces bayanus (used in winemaking and cider production), and Saccharomyces pastorianus (used in lager beer brewing) [4].
Ale, lager, porter, stout, lambic, weiss, and many other denominations accompany the term "beer" to indicate specific brewery products with unique organoleptic and chemical properties.
The diversity of beers has been obtained because producers have often maintained local traditions which have yielded products with distinctive characteristics linked to the native territory [5]. In recent years, the diversity of products has increased with the rise of smaller breweries creating bespoke beers. This worldwide economic phenomenon has been called the "craft beer revolution" [6][7][8]. Initially, only two types of yeasts were used, selecting them according to the ability to flocculate: top fermenting (ale yeast) [9] and bottom fermenting (lager yeast) [4].
Currently, the main criterion accepted for beer classification relies on the brewing process, which divides it into three macro-categories: ale, lager, and lambic beers. Ale beer is brewed by using S. cerevisiae strains, while lager-style beer involves S. pastorianus strains, and lambic-style beer is obtained from spontaneous fermentation of indigenous yeasts present in the raw materials used [10].
Today, modern brewers tend to use new ingredients, including spices, herbs, and fruits, to enhance the flavor. However, yeasts still have the main role in defining the organoleptic characteristics of beer [11].
Up until a few years ago, the selection of starter yeasts, used in beer production, was exclusively carried out on strains belonging to the genus Saccharomyces; this is because these yeasts were predominant in spontaneous fermentations [2,12,13].
In contrast, non-Saccharomyces yeasts have often been ignored, both because they are not very predominant in the fermentation process and also because of their high production of off-flavor compounds, such as acetic acid, diacetyl, and 2,3-butanediol [14,15]. Despite this, the rising demand of new specialty beers has driven researchers to isolate and reevaluate the potential benefits of non-Saccharomyces yeasts in beer production.
Non-Saccharomyces yeasts possess, unlike Saccharomyces yeasts, many enzymatic activities which can lead to the production of metabolites that contribute to a greater aromatic complexity of alcoholic beverages [16][17][18][19].
Michel et al. [11] and Basso et al. [12] emphasized the great potential of non-Saccharomyces yeasts to develop beers with different alcohol contents and a broad range of flavors. They highlighted the varying abilities of unconventional yeasts to metabolize desirable aroma-active substances, such as fruity esters, monoterpenes, higher alcohols, phenols, and acids.
In beer, the production of flavor-active compounds is strictly strain-dependent [20]. Thus, the choice of yeast strains used in beer production is essential to obtain products with desirable and distinctive sensory properties.
In the beer sector, the genus Saccharomyces can traditionally be divided into two groups: ale and lager yeasts, also known as the top-fermenting and bottom-fermenting yeasts, respectively [23]. This differentiation was originally made because the strains were classified on the basis of their flocculation property. S. cerevisiae traditionally conducts "top fermentation" where yeasts aggregate on the surface of the fermenting wort.
Traditionally, S. pastorianus, hybrids of S. cerevisiae and S. bayanus [4], is the yeast used for lager-style beer fermentations. This facilitates the harvesting and cropping of yeasts from fermented must and can be used as a starter in subsequent fermentations [3,24].
Lager represents almost 90% of the beer market; ale beer represents 5% of the beer market [15]. The remaining percentage is taken by beers produced by spontaneous fermentation with indigenous yeasts and bacteria [25,26]. Ale beers are brewed by S. cerevisiae strains at a fermentation temperature of 15-25 • C, while lager-style beer involves S. pastorianus strains in a process conducted at a temperature of 8-12 • C [1]. In all cases, a positive result depends on the choice of the yeast strain used and its vigor and vitality.
The process control parameters, such as sufficient nutrient supply, correct inoculation (pitching) rate, optimized dissolved oxygen addition, and temperature control, are important for proper yeast metabolic activity and for beer quality [27].
The production of quality beer relies on the activity of fermenting yeasts that are qualified not only for good fermentation yield efficiency, but also for affecting aroma and flavor of the beverage.
The volatile part of the beer includes over 800 different compounds, but only some of them are known to be active for flavor [28,29]. A large part of these compounds, dissimilar to the aromatic compounds present in malt and hops, are synthesized during alcoholic fermentation and have an important impact on the aroma and taste of beer [30].
In Table 1, some classes of metabolites produced by Saccharomyces yeasts during alcoholic fermentation, the main compounds that characterize the classes, and their principal influence on the sensorial characteristics of the beers have been reported.
The production and quantity of these compounds depend on the yeast strains involved in the fermentation process and, therefore, their choice is of fundamental importance [3,[30][31][32][33][34][35][36][37][38][39]. In addition to the traditional S. pastorianus and S. bayanus natural hybrids strains [4,40] that have been used extensively by the brewing industry [23], the recent discovery of S. eubayanus [41] has allowed the creation of brewing yeast hybrids generated by de novo hybridization [42][43][44][45][46][47][48]. Excessive concentrations of acetaldehyde carbonyl compounds cause stale flavor in beer and impart an undesirable "cut grass" or "green apple" character. Diacetyl contributes negatively with a buttery flavor to the beer [49,51] Organic acids Succinic, citric, acetic, malic and pyruvic acids The balance between sourness and sweetness of a beer is of great importance. [52,53] Polyols Glycerol Contribute to the smoothness [49] Sulphur compounds Hydrogen sulphide, Dimethyl sulphide, Sulphur dioxide, Thiols Small amounts of sulphur compounds can be acceptable, or even desirable in some beers, in excess they give rise to unpleasant off-flavors. [49,50] Saccharomyces interspecific hybrids, both artificial and natural, have a significant potential in industry. This is because they, compared to parents, often show the synergistic phenomenon of heterosis, also called hybrid vigor, which is the tendency to outperform parents in fermentative performances. [47,48,54]. In this context, the hybridization of Saccharomyces brewing yeasts, besides improving fermentation efficiency, also offers the possibility of generating novel non-genetically modified (non-GM) strains with unique properties [47,55]. In fact, many studies on yeast hybrids have focused on attempting to increase the formation and diversity of aroma-active compounds in beer. Table 2 shows studies published on the use of Saccharomyces hybrids in brewing.
These studies have been mainly limited to hybrids created with S. cerevisiae, S. bayanus, and S. eubayanus strains as parents. Many other species in the Saccharomyces genus possess traits desirable for brewing, including cold tolerance and high ester formation, and thus represent feasible alternatives to S. eubayanus in interspecific hybrids for lager brewing purposes [48,56]. Table 2. Studies published on the use of Saccharomyces hybrids in brewing.

Species Comments References
S. cerevisiae ale/S. cerevisiae sake Beer brewed using hybrids contained more ethanol and esters compared to beer brewed using the parent strains. [57] S. cerevisiae ale/S. cerevisiae (syn S. cerevisiae var. diastaticus) Hybrid had higher osmotolerance and higher ethanol yield than the parent strain. [58] S. cerevisiae ale/Cold-tolerant S. bayanus Hybridization with S. bayanus is useful to improve low-temperature fermentability of the top-fermenting yeast S. cerevisiae. [59] S. cerevisiae ale/S. bayanus Hybrid strains, compared to the lager parent strain, showed improved stress resistance (osmo-and temperature tolerance), fermentation performance and improved survival at the end of fermentation. [60] S. cerevisiae, S. paradoxus, and S. pastorianus Some hybrids show a distinct heterosis (hybrid vigor) effect and produce greater quantities of isoamyl acetate than the best parental strains, while retaining their overall fermentation performance. [61] S. cerevisiae/S. eubayanus The hybrid had improved tolerance to low temperatures and the capacity of oligosaccharide utilization, compared to the parent strains. [43] S. cerevisiae/S. eubayanus Hybrids inherited beneficial properties from both parent strains (cryotolerance, maltotriose utilization and strong flocculation) and showed apparent hybrid vigor, fermenting faster and producing beer with higher alcohol content than the parent strains. [44] S. cerevisiae ale and wine strains/S. eubayanus Hybrids produced a greater diversity of aroma compounds compared to traditional lager yeast and parent strains. [46] S. cerevisiae ale strain/S. eubayanus type strain Some hybrids showed increased fermentation rates and produced higher concentration of flavor-active esters. [45] S.cerevisiae/S.uvarum The hybrid strain possesses a range of industrially desirable phenotypic properties, including broad temperature tolerance, good ethanol tolerance, and efficient carbohydrate use. [56]

Non-Saccharomyces Yeasts in the Brewing Process
It is generally assumed that non-Saccharomyces yeasts play a negative role in the brewing process resulting in problems associated with beer turbidity, filterability, viscosity, phenolic off-flavors, and other negative flavor profile changes [39]. However, in recent years, the selection and use of non-Saccharomyces yeasts has been a biotechnological resource in brewing [62,63]. In fact, in some cases, the use of these yeasts allows, in addition to some technological advantages, also an improvement and diversification of the sensory profile of the beers [12,64]. For example, the characteristic lambic beer sensory profile is caused by spontaneous fermentations of non-Saccharomyces yeasts, including, in particular, Brettanomyces bruxellensis strains. These yeasts, besides having a high volatile acidity, also produce esters, such as ethyl acetate, ethyl caprate, ethyl caprylate, and ethyl lactate, which characterize the typical sour flavor of lambic beer [10,[65][66][67][68].
Non-Saccharomyces yeasts have low primary metabolic efficiency, which end up having a limited fermentation performance. However, compared to Saccharomyces, they possess greater enzymatic capacities and metabolic pathways for the synthesis of volatile compounds that contribute to the flavor and aromatic profile of the final product [12,[17][18][19]69,70].
In addition to the compounds which are formed during the alcoholic fermentation, monoterpene alcohols (linalool, α-terpineol, β-citronellol, geraniol, and nerol) derived from hops contribute to the sensory characteristics of beer. In wort, these compounds are often present in glycosidically bound forms and aromatically inactive [71]. Some non-Saccharomyces yeasts have beta-glucosidase, an important enzyme for the hydrolysis of glycoconjugate precursors and the release of active aromatic compounds [72].
With the spread of the microbrewing phenomenon (craft revolution), in recent years, there has been a growing interest in innovation and production of beers with greater sensory complexity. The selection and use of unconventional yeasts allow the production of distinctive products that are not included in the traditional beer on offer and, at the same time, can satisfy new consumer trends. [65].
The effect of pure or mixed starters, using Saccharomyces and non-Saccharomyces strains in beer production, has also been examined [26,35]. Some metabolic products and their main impacts on beer composition of non-Saccharomyces starter are summarized in Table 3.
However, unlike the wine sector, where non-Saccharomyces commercial starters (Torulaspora delbrueckii, Metschnikowia pulcherrima, Metschnikowia fructicola, Kluyveromyces thermotolerans, Kluyveromyces wickerhamii, Candida zemplinina, Schizosaccharomyces pombe, and Pichia kluyveri) are now widely used for innovative applications [78], the commercial offer in the beer sector is very limited. In fact, of all the yeasts mentioned, only a few strains belonging to the genus Brettanomyces spp. are available as non-Saccharomyces commercial starters. Due to the limited supply, a recent study assessed the effect of non-Saccharomyces yeast strains selected and marketed for the wine sector, in beer fermentation [79]. In breweries and wineries, Brettanomyces are typically recognized as spoilage yeasts, being the cause of major economic losses. Its presence can completely change the organoleptic properties of the product, creating a controversial characteristic, which is mainly due to the production of secondary metabolites when performing alcoholic fermentation. These metabolites have been associated with undesirable flavors, depicted as horse sweat, barnyard, medicinal, or leathery. However, used appropriately, Brettanomyces can contribute to fruit flavors (e.g., pineapple, mango, pear, grape, lemon) and, today, they are often used in craft beers. Over the last decade, the craft beer sector has constantly demanded novel attractive flavors, and there has been a rising interest in understanding Brettanomyces species, exploiting its potential in beer fermentation [66,67,80,81]. The species most commonly marketed for the beer sector belongs to the B. bruxellensis species, which is proposed as a pure crop or in mixed cultures with B. lambicus, B. anomalus, B. claussenii, B. nanus, B. naardenensis, and B. custersianus or with Lactobacillus and Pediococcus strains for sour beer production [82]. These cultures also produce large amounts of ethyl lactate and ethyl acetate, along with some acetic acid, but the commercial success is mainly due to their high β-glucosidase activity that allows the release of monoterpene aromatic alcohols from hops, enhancing "flowery" and "citrus" characteristics in the beer.

Low-Alcohol Beer
Over the last years, the consumption of alcohol-free beer has risen significantly due to the fact that it represents an alternative to standard soft drinks. The expansion of the beer industry into new markets (e.g., beer production in countries where alcohol consumption is banned) could motivate researchers to look for new strategies to reduce alcohol content in beer. There is an increasing interest of consumers in health issues, and no alcohol policies for drivers and pregnant women [110]. Beers with reduced alcohol content are often classified as "low-alcohol" beers containing 1.2% v/v of ethanol, and "non-alcohol" or "alcohol-free" beers containing 0.5% v/v of ethanol [113]. However, most of the nonalcoholic beers available have a flavor profile that is not well accepted [114][115][116]. These beers can be produced through a physical process, involving ethanol removal from beer, or a biological process, based on limited ethanol formation during beer fermentation [117]. Physical methods include: vacuum rectification and evaporation, spinning cone column distillation, osmotic distillation, dialysis, and reverse osmosis [118,119]. This method requires considerable investments in the special equipment for alcohol removal, and the final beer is characterized by poor sensory qualities caused by losing higher alcohols and esters [96]. The aim of the other method, often referred to as biological, is to reduce alcohol production during the fermentation process. In general, in the biological processes of obtaining low alcoholic beer, the wort is not completely fermented, either with the interruption of fermentation, cold fermentation, immobilization of yeast, or use of nonconventional yeast [113,[120][121][122][123][124]. The great challenge presented to all breweries that produce non-alcoholic beers is to develop products that have organoleptic characteristics that are as close as possible to the original beers and, thus, present a greater acceptance by consumers [114]. In this sense, non-conventional yeasts, unable to utilize maltose and/or maltotriose, represent a very interesting alternative to classical physical methods, both for the lower production costs and for the advantage of producing low-alcohol beers with an aromatic complexity similar to that of standard beers [12,55,75,113]. An example of this is the use of S. ludwigii in brewing [97,98,125]. Beer produced with this yeast contains very low alcohol concentration and shows high amounts of esters that contribute to a "fruity" characteristic [98]. Similarly, beer brewed with strains of P. kluyveri and T. delbrueckii that cannot degrade maltose could be an interesting way to produce beers with a lower alcohol content but rich in flavor and fruity aroma. In Table 4, some non-Saccharomyces species tested for low-alcohol beer production with the relative references are reported. Beers produced using this strategy contained a more complex flavor profile compared to single fermentations with S. cerevisiae and, in some cases, lower alcohol content [87,126].
Recently, gene modification (GM) approaches have been used to generate a yeast strain able to produce a low alcohol beer with rich fruity flavor and aroma. Considering the negative public perception toward genetically modified organisms (GMO) and that the legislation in various countries restricts the sale of products containing either GMO or GMO in their manufacture, no GM approaches have been developed for the generation of beer yeasts that produce less ethanol. Adaptive evolution offers a potential no GM strategy to generate yeast strains that produce reduced amounts of ethanol [127]. This approach relies on applying a selection pressure that favors a metabolic diversion away from ethanol production. Several reagents can be used to shift yeast carbon metabolism and potentially generate "low alcohol" strains [120]. It is possible that a selection pressure that favors the survival of individuals with enhanced glycerol production will also lead to adaptations resulting in lower ethanol production [128]. However, the results obtained so far suggest that the generation of "low-ethanol" strains by adaptive evolution is not an easy task, and that promising strain isolates may need further improvements in order to create a marketable yeast product. [113]. Table 4. Non-conventional yeasts in low alcohol beer production.

Probiotic Beer
Functional beers are obtained by enrichment with health-promoting substances, therefore, they are considered as beers giving health benefits, if consumed in moderate amounts. Among the functional beers, an absolute novelty is represented by probiotic beer, obtained by incorporating probiotic microorganisms [132]. Craft beer, which is unpasteurized and unfiltered, can be considered as a new tool to provide beneficial health effects. In contrast, the technologies applied to pasteurized or filtered beers are not suitable for this purpose, as heat or filtration can kill or remove the probiotics, unless the addition of probiotics takes place after pasteurization or filtration. Therefore, because viability is crucial for the effectiveness of probiotics, it could be more suitable to produce craft beer as a probiotic beer, rather than an industrial beer [132]. Most probiotic microorganisms are bacteria, whereas Saccharomyces cerevisiae var. boulardii (synonym S. boulardii) is the only non-conventional yeast used extensively as a probiotic and often marketed as a dietary supplement [133,134]. S. boulardii possesses many properties that make it a potential probiotic agent: the survival at body temperature (37 • C), the resistance to stomach acids and bile acids, and the survival to the competitive environment of the intestinal tract [135,136]. This yeast, isolated from fruit in Indochina [137], has shown a capability to prevent infectious diarrhea usually caused by bacteria, such as Escherichia coli, and to inhibit invasive properties of Salmonella typhimurium. In addition, it has been very effective against Clostridium difficile in the prevention and treatment of antibiotic-associated diarrhea [138] and may help to eradicate Helicobacter pylori [139]. To present, it is the only yeast species available on the market with probiotic properties.
In recent studies, S. boulardii has had suitable resistance to alcohol and gastrointestinal conditions for probiotic alcoholic beverage development [140]. The use of S. boulardii as a mixed starter with S. cerevisiae for craft beer, or as single starter for alcohol-free beer production has been proposed recently [141][142][143]. In other studies, S. boulardii, used as a single yeast starter culture for brewing, has been shown to produce craft beer with higher antioxidant activity, lower alcohol content, and similar sensory attributes as the craft beer obtained with S. cerevisiae strains. [132,144]. Within this framework, the beer is likely to become a new medium for successful release of probiotic microorganisms, but further research is needed to test the shelf life.
In this type of product, as in other types of unfiltered beers, the presence of yeasts gives the beer its unique taste, but greatly reduces its shelf life, generally to no more than two months; the unfiltered beers are then marketed directly in microbreweries or nearby. In the storage of unfiltered beers, there are changes that, after yeast lysis, can negatively affect its quality (e.g., off-flavor compound production) and probiotic properties. Therefore, in the production of unfiltered beers, such as a probiotic beer, the selection of the yeast strain to be used is very important and must also be studied according to the intracellular substances that are released during the storage of beer, such as antimicrobial and antioxidant compounds.
Following the considerations, future studies exploring the isolation of novel brewing yeasts and their potential application in beer probiotication could open interesting biotechnological innovations and new perspectives.

Conclusions
This study has not only given an overview of the Saccharomyces yeasts normally used in beer production, but also future prospects that biotechnological research could offer in the enhancement of this ultra-millennial drink. The microbrewing phenomenon (craft revolution) and the growing demand for specialty beers has stimulated researchers and brewers to select new yeast strains possessing specific metabolic properties. In particular, the use of non-conventional yeasts can allow the production of innovative products, which are not included in the traditional beer offer, and, at the same time, following new market trends to meet consumer demands.