Influence of Wort Composition and Fermentation Parameters on Metabolic Activity of Non-Saccharomyces Yeast in Non-Alcoholic and Low-Alcohol Brewing

Abstract

As consumer attitudes shift, non-alcoholic and low-alcohol beers (NABLABs) have grown rapidly in popularity. This has driven interest in biological production methods that avoid the cost and flavor damage associated with post-fermentation dealcoholization. This review focuses on how barley wort composition and process conditions shape the metabolism of maltose- and maltotriose-negative non-Saccharomyces yeasts (NSYs), and how this, in turn, affects ethanol yield, flavor, and aroma in NABLABs. Key sections examine differences in carbohydrate utilization between Saccharomyces and NSYs, the influence of oxygen and Crabtree/Kluyver effects on carbon flux, and the roles of glycerol and organic acid formation as alternate carbon sinks that also contribute to mouthfeel, sweetness perception, and acidity. Particular attention is given to mashing strategies and enzyme additions used to redesign wort sugar profiles for NSYs, including high-temperature, low-gravity mashes and exogenous amyloglucosidase to increase glucose while limiting maltose and ethanol formation. The review also summarizes how the NSY-driven production of esters, higher alcohols, and the biotransformation of hop-derived precursors can offset excessive sweetness and “worty” off-flavors that commonly affect NABLABs. The use of NSYs opens an exciting array of opportunities for brewers to make NABLABs; however, challenges remain. Saccharomyces yeasts have centuries of brewing experience behind them and the adaptations needed for effective use of NSYs are still in development. Fundamentally, the challenge for NABLAB brewers using biological methods is to balance the desirable effects of fermentation while maintaining ethanol levels below the target threshold. This review outlines those challenges in detail and examines some of the approaches that are being used to solve them.

1. Introduction

Beer has been one of the most consumed beverages globally and a social aspect of celebrations throughout history. Dating back to 8000 BCE, fermentation represents the earliest documented form of biotechnology, using spontaneous fermentation to make low alcohol beverages [1]. While subsequent advancements have led to the production of higher-alcohol beers, over the past decade, there has been a reversion to beer’s low-alcohol roots due to an increased demand for non-alcoholic and low-alcohol beer (NABLAB). There are numerous drivers of this increased demand, including generational shifts in drinking preferences, athletes, pregnant women, religious beliefs, health concerns, alcohol intolerance, or general lifestyle choices. Although this rapid growth is a modern phenomenon, low-alcohol beer has existed throughout history. “Near beer” was introduced as a response to the prohibition era in the USA from 1920 to 1933, with a limit of 0.5% alcohol by volume (ABV) set by the Volstead Act. This resulted in major American brewer introducing and producing non-alcoholic counterparts to their flagship products [2]. Similarly, Germany had its own version of non-alcoholic beer called “vitamalz” or “kinderbier”, brewed similar to beer but with low or no fermentation. This pattern has resurfaced as consumers become more health-conscious. However, due to advancements in biotechnology, the methods used to produce NABLABs are more sophisticated and efficient [1]. The definition of what can be considered a NABLAB varies depending on the country. There are also different terms used to describe these beverages, such as “alcohol-free beer,” “near beer,” “low-alcohol beer”, or “alcohol-reduced beer” Most EU countries, like Germany and Belgium, consider alcohol-free beer to be >0.5% ABV, while the USA and the Netherlands have stricter limits of 0–0.1% ABV [3,4]. In terms of low-alcohol beer, definitions vary more widely, ranging from 1.2% to 2.8% ABV [5]. The brewing process consists of the following steps: malting, milling, mashing, boiling, cooling, fermentation, maturation, filtration, carbonation, microbiological stabilization, and packaging. The various NABLAB production techniques follow a similar process but include strategies to either remove ethanol or produce less of it during fermentation [1,6]. Recent studies have also indicated the importance of the mashing process and fermentable sugar production to produce NABLABs [5,7]. Fermentation is a key step in brewing, ultimately deciding both the ethanol content produced by the yeast as well as the flavor profile including desirable-volatile-compound production [8,9,10]. While previous reviews have described the different non-Saccharomyces yeasts (NSYs) used in the production of NABLABs and their flavor profiles [11,12,13], there is a need to define and summarize the interaction between NSY metabolism and brewing parameters as more research is being produced on the subject. This will help us understand the effect of changes in brewing practices on the metabolism of NSYs and consequently make the outcomes more predictable. This review summarizes the current knowledge of how wort composition affects NSY metabolism in NABLAB brewing. This includes analysis of the fate of carbon in NSYs currently used in brewing and how it impacts the sweetness and aroma of the resulting NABLABs.

2. NABLAB Production Methods

There are many methods to produce NABLABs (Figure 1), which are broadly divided into two categories: physical (removal of ethanol post fermentation) and biological (controlled ethanol production) [12].

2.1. Physical Methods: Post-Fermentation Ethanol Removal

Physical methods such as thermal evaporation and membrane separation are used to remove ethanol from already-produced beer [5,9,12,14]. The removal process using these methods needs to be optimized further in order to preserve the sensorial quality of the produced NAB. A key advantage of this method is its ability to achieve 0.0–0.5% ABV, with alcohol virtually undetectable.

There have been significant developments in thermal evaporation processes for removing ethanol while retaining desirable beer volatiles. One of the most effective ways is vacuum rectification, which consists of preheating the beer in a plate heat exchanger, degassing the beer to simultaneously liberate volatile compounds that are later recovered, and dealcoholizing in a vacuum column [12,15]. This process can remove virtually all alcohol from beer; however, it poses the risks of thermal damage or volatile loss from the beer [14,15,16].

Another type of physical removal of ethanol from NABLABs includes membrane processes, which exploit the semipermeable nature of membranes to separate smaller molecules, such as ethanol and water, from the beer into the permeate liquid. One such process is reverse osmosis, in which beer flows tangentially to the membrane surface, and ethanol permeates the membrane selectively when the transmembrane pressure exceeds the beer’s osmotic pressure [12,17]. While it is assumed that this will remove ethanol and retain aroma and flavor compounds, this has not been the case in practice as many flavor-active molecules have low molecular weights similar to or even lower than ethanol [18]. The available data show a significant loss of volatiles due to the imperfect selectivity of membranes [16]. This issue could be addressed by increasing transmembrane pressure, leading to a higher rejection of ethanol and higher alcohols but lower rejection of esters.

The production costs for the current state of NABLAB brewing remain high due to sophisticated equipment for physical or chemical alcohol removal, as well as energy and personnel costs. Moreover, smaller breweries do not have the ability to incorporate these techniques into their production lines due to the inaccessibility of special equipment, which requires high initial investment in funds and personnel [12,13]. Moreover, these methods often compromise volatile retention along with the removal of ethanol, requiring additional process optimization and/or post-process flavor correction.

2.2. Biological Methods: Controlling Ethanol Formation

Biological methods include arrested fermentation, cold-contact fermentation, and the utilization of specialized yeasts such as NSYs (

Table 1. Comparison of commonly used NSY species in NABLABs, brewing strategies, and fermentation performance.

Non-Saccharomyces Yeast	Unique Features	Experimental Approach	Parameters Measured	Potential Carbon Sinks	Source
Lachancea fermentati	Acidification increases sourness and lowers pH to 3.61	Pilsner barley malt mash: 20 min-50 °C, 60 min-65 °C, 5 min-78 °C; O.G. ~6.6 °P; Magnum hop pellet at 10.4 IBU; pitch rate ~8 × 106 cells/mL; pH 5.44 Fermentation temperature: 25 ± 1 °C (uncontrolled); duration: 36 h	ABV%, residual sugars, pH, organic acids, FAN, volatile compounds, sensory analysis	2.1% ABV, lactic acid, glycerol	[21]
Pichia kluyveri	Esters (Ethyl butyrate; isoamyl acetate) + glycerol, aroma and mouthfeel; restricted sugar use	Two-row spring barley malt mash 60 min: 72 °C-10 min-78 °C; O.G. 6.5 °P; Magnum hop pellets type 90; 20 EBU; pitch rate ~2 × 105 CFU/mL; pH 6.17; fermentation temperature: 20 °C Duration: 6 days	ABV%, residual sugars, pH, glycerol, organic acids, FAN, volatile compounds, dimethyl sulfide, sensory analysis, dimethyl sulfide	0.12–0.26% ABV, glycerol, esters	[10]
Saccharomycodes ludwigii	Low-alcohol due to restricted maltose use; esters and higher alcohol significant but variable with strong strain-to-strain differences	Pilsner barley malt high-temperature mash: 76 °C for 90 min; O.G. 12 °P; pitch rate ~106 cells/mL; fermentation temperature: 20 °C; pH ~5.4; duration: 10 days	ABV%, residual sugars, pH, volatile compounds	0.93–3.32% ABV, high-residual sugars, esters	[22]
Cyberlindera saturnus	“fruity”, pear, maracuja, mango, aroma; (Ethyl acetate, Isoamyl acetate); glycosidic precursor (E)-β-damascenone	Spray-dried barley malt extract; O.G. 6.97 °P; unhopped; pitch rate ~5 × 106 cells/mL; fermentation temperature: 16.1 °C; pH 5.2; duration: based on CO2 production	ABV%, attenuation, residual sugars, pH, organic acids, volatile compounds, sensory analysis	~0.60% ABV; high-residual sugar, esters	[23]
Metschnikowia pulcherrima	Moderate production of polyphenol; no ethyl acetate	Brewers spent grain from PILS wort at 78 °C; O.G. 3.3 °P; cascade hops at 20 IBU; pitch rate ~3 × 106 cells/mL; fermentation temperature: 20 °C; pH 5.5; duration: based on CO2 production	ABV%, attenuation, residual sugars, pH, FAN, volatile compounds, sensory analysis	~0.23% ABV; lactic acid	[24]
Hanseniaspora uvarum	high glycerol production	Pale ale malted barley: AMG added + glucose-dominant wort; O.G. 7 °P; iso-alpha acid extract; pitch rate ~107 cells/mL; fermentation temperature: 25 °C; pH 5.3; duration: 7 days	ABV%, residual sugars, pH, glycerol, organic acids, biomass	High-residual sugars, glycerol	[25]
Saccharomycopsis fibuligera	Plum/berry, dried fruit, maltose transporter present	Sterilized barley malt extract; O.G. 10.2 °P; unhopped wort; pitch rate ~107 cells/mL; fermentation temperature: 20 °C; pH 5.3; duration: 20 days	ABV%, attenuation, residual sugars, pH, sensory analysis	~0.83–1.20% ABV, high-residual sugars	[26]
Mrakia gelida	VDK control, apricot, grape and litchi	Pale malt step mash: 68 °C-60 min, 71 °C-10 min, 78 °C-10 min; O.G. 12 °P; Hallertau Magnum hops at 25 IBU; pitch rate ~107 cells/mL; fermentation temperature: 10 °C; pH 5.44; primary fermentation duration: 22 days	ABV%, attenuation, residual sugars, pH, FAN, volatile compounds, sensory analysis	~1.40% ABV, high-residual sugars	[27]
Kazachstania servazzii	Pear, apple notes; 3-methylbutyl acetate	Pilsner malt step mash: Mashing-in at 48 °C; temperature steps: 48 °C 30 min-63 °C 30 min-72 °C 30 min-78 °C 10 min; O.G. 12 °P+ dilution 8 °P; Magnum hops at 40 IBU; pitch rate ~107 CFU/mL; fermentation temperature: 25 °C; pH 5.1; duration: 6 days	ABV%, attenuation, pH, volatile compounds, sensory analysis	~0.73% ABV, high-residual sugars, esters	[28]

) [19,20]. The fermentation process is a crucial step in brewing that determines the trajectory of volatile production. Methods like arrested fermentation or cold-contact fermentation aim to reduce ethanol production during the fermentation process.

In arrested fermentation, the yeasts are allowed to ferment the wort partially and then are removed from the media, thus stopping fermentation. Thus, the brewer intentionally creates an incomplete fermentation. However, the resultant beer is poor in aromatic compounds and high in worty character, generally remedied by externally added aroma compounds. At the same time, the incomplete fermentation does not provide the yeast with enough time to reduce off-flavor compounds like diacetyl [12,29,30].

Alternatively, cold-contact fermentation limits yeast metabolism by using low temperatures and short fermentation times. This method produces higher levels of volatile compounds than arrested fermentation but has a lower capacity to reduce the aldehyde compounds responsible for worty off flavors (e.g., methanal). Additionally, at low temperatures, polyphenols in the wort can bind to aldehydes inhibiting yeast-mediated reduction. Polyphenols are also well documented to interact with haze-active proteins and hence contribute to colloidal instability. Some attempts have been made to improve this process such as using genetically modified yeast to produce low ethanol at a higher fermentation temperature to help reduce the aldehydes or by using polyvinylpolypyrrolidone (PVPP) after wort cooling to remove polyphenols [8,19,29].

An alternative biological strategy for NABLAB brewing is to use maltose-negative yeast, whether they be non-Saccharomyces yeasts (NSYs) or genetically modified maltose-negative Saccharomyces yeasts [10,24,28,31]. The maltose-negative yeasts are unable to ferment the dominant sugar present in traditional barley worts and thus produce lower ethanol compared to traditional brewer’s yeast. This method of NABLAB production is similar to manufacturing standard beer while also producing unique and potentially desirable aroma and mouthfeel characteristics from this non-traditional yeast. However, there are potential tradeoffs such as excess sweetness [6,13,32,33,34].

3. Advantages and Disadvantages of Non-Saccharomyces Yeasts in NABLAB

Non-Saccharomyces yeasts have been studied broadly in wine fermentations in sequential or co-inoculation with Saccharomyces cerevisiae [35,36,37]. While yeasts such as Brettanomyces are considered spoilage organisms in wine, their controlled use has proved to be effective in reducing ethanol and producing desirable flavors, increasing acidity, and enhancing mouthfeel [38,39,40,41]. They seem to be a promising alternative in NABLAB production due to their availability, low-cost potential, and ability to improve aroma complexity [11,20]. Most of the NSYs widely studied for NABLAB production are associated with wine and are therefore more adapted to grape must sugars like glucose and fructose rather than maltose and maltotriose [42,43]. Other yeast sources from cider, kombucha, and sourdough have the potential to be repurposed for NABLAB production [28,31,44]. Unlike winemaking and cider production, where the main fermenting yeast Saccharomyces cerevisiae is co-inoculated/sequentially inoculated by wild-type NSYs [45,46,47], brewing typically utilizes NSYs in pure culture after boiling and aseptically cooling the wort [10,39,48]. However, there could be interesting applications of combining different low-ethanol-producing NSYs [49].

NSYs, such as Hanseniaspora uvarum and Pichia kluyveri, are reported to produce a high concentration of esters such as ethyl acetate, ethyl hexanoate, ethyl dodecanoate, and isoamyl acetate, providing a fruity aroma to wine and beer [40,50]. They not only reduce the ethanol but also introduce complex flavor profiles in beer. Apart from aroma compounds, they are also capable of producing organic acids and glycerol, contributing substantially to beer body and taste, which may be particularly important for low-gravity NABLABs [51]. These metabolites are produced by NSYs under microaerobic conditions in which carbon is channeled away from ethanol production towards other metabolites such as organic acids, glycerol, and biomass [32]. The ability of certain NSYs to produce extracellular hydrolytic enzymes may play an important role in aroma production [52,53]. This is due to their ability to produce high extracellular β-glucosidase, an important enzyme for the hydrolysis of glycoconjugate precursors present in hops, resulting in the release of monoterpene alcohols (linalool, α-terpineol, β-citronellol, geraniol, and nerol) [52,54,55].

Due to their maltose-negative nature, however, some NSYs are unable to use most of the fermentable sugars in the form of maltose or maltotriose present in barley malt wort. This impaired maltose and maltotriose utilization by NSYs produces NABLABs with high amounts of residual maltose and dextrins [43]. Besides the sensory implications, the high concentration of residual maltose also poses a high risk for microbial contamination [56,57]. This demands a highly controlled and hygienic post-fermentation processing environment to prevent refermentation and spoilage. Moreover, the maltose utilization capabilities of NSYs are species- and strain-dependent [21,22,28]. The further study of the differences in yeast metabolism in the context of brewing is therefore important to understand the patterns in metabolite production under different fermentation parameters. Commonly studied NSYs include Saccharomycodes ludwigii, Torulaspora delbrueckii, Lachancea thermotolerans, Pichia kluyveri, Kluyveromyces marxianus, Zygosaccharomyces rouxii, Wickerhamomyces anomalus, Mrakia gelida, Hanseniaspora uvarum, and Kazachstania servazii. This list continues to grow as more species are tested for their NABLAB abilities [24,27,30,31,48,58,59].

In the process of yeast screening for use in NABLABs, certain key aspects need to be taken into consideration, such as their resistance to hop-derived iso acids, their ability to flocculate, and what metabolites they can consume in anaerobic conditions [31]. It is crucial to perform these initial screenings for NSYs due to their high variability depending on both strain and species [33]. Another important aspect is the fermentation kinetics of the yeast in wort media under anaerobic conditions [60]. This gives us information about the rate of sugar consumption and fermentation completion, often indicated by CO₂ production or residual sugar concentrations, giving us further control over ethanol production [60]. While there have been a variety of parameters affecting ethanol and aroma production by different NSYs at the species and strain levels, there are still certain species that are more promising in the reduction of ethanol due to their inability to efficiently utilize fermentable sugars in the wort and aroma production due to high enzyme activity. Some examples are Pichia kluyveri [10,28,32,35,40,49,61,62,63,64], known for producing highly fruity esters, Lachancea thermotolerans, for producing distinct amounts of lactic acid [65,66], and Cyberlindera saturnus, for its production of cool mint and stone fruit characteristics [23,67].

The performance of both Saccharomyces and non-Saccharomyces yeasts will be explained in detail in terms of carbohydrate utilization, oxygen effects on yeast metabolism, and different carbon sinks for the carbohydrates utilized, namely, ethanol, glycerol, organic acids, and flavor production [65,66].

4. Yeast Metabolism in the Context of Barley Wort Composition

4.1. Carbohydrate Utilization by Saccharomyces vs. Non-Saccharomyces Yeast

Differences in yeast metabolism can have important impacts on aroma and flavor in NABLABs. Saccharomyces cerevisiae is commonly used in the fermentation of wine, bread, cider, beer, and sake. Properties such as ethanol production, tolerance to ethanol and hop acids, and preference for fermentation over respiration in the presence of high sugar concentrations have made domesticated S. cerevisiae strains an ideal for controlled fermentations in breweries across the globe [65,68,69]. Yeast strain selection and wort composition play an important role in the flavor and aroma development in beer [70,71]. Yeast cells utilize nutrients present in the wort for growth and fermentation through metabolic pathways (e.g., glycolysis, TCA cycle, etc.) [72,73]. Fermentation begins with the breakdown of glucose to pyruvate through the glycolytic pathway, followed by conversion to acetaldehyde and then to ethanol with the concomitant production of carbon dioxide. This process results in both the formation of two ATP molecules from glycolysis as well as the regeneration of 2 NAD+, necessary to maintain the glycolytic pathway (Figure 2) [65].

Saccharomyces cerevisiae is efficient in utilizing maltose, maltotriose, glucose, fructose, and sucrose, converting them primarily to ethanol both under anaerobic and aerobic conditions [74,75,76]. On the other hand, NSYs only use glucose, fructose, and sucrose for fermentation, having a limited ability to use maltose, the dominant fermentable sugar (~50–80%) in barley wort [20,24,77,78]. This limited fermentation of the majority fermentable sugar limits ethanol production, but at the cost of leaving high amounts of residual maltose in the final NABLAB product [6,13,39]. In Saccharomyces, glucose and fructose are the first to be metabolized by the cell by passive facilitated diffusion, which does not require energy [79]. Maltose and maltotriose utilization requires active transport with the help of the following: (1) an energy-dependent maltose permease and (2) maltose-hydrolyzing enzymes in the cytoplasm converting them to glucose (e.g., amyloglucosidase/glucoamylase) [65,80]. Maltose consumption is also inhibited by glucose concentrations greater than 1% w/v through catabolite repression mechanisms [79]. Among most NSYs, it is unclear whether the reason for the lack of maltose utilization is the same in all cases; however, the glucoamylase enzyme from S. cerevisiae is widely conserved across these NSY species, suggesting that maltose transport is the limiting factor [81]. Some studies have reported the growth of NSYs in maltose media under aerobic conditions, during which they could produce sufficient energy for growth even with limited uptake [82]. For efficient fermentation to occur, yeast cells must be able to grow in wort and assimilate fermentable sugars and key nutrients drive the formation of flavor-active compounds that shape beer aroma and taste [83,84]. The molecular and physiological characteristics of the yeast strain used are key determinants of fermentation performance, with the main bottleneck being the introduction of sugars into the cytoplasm of the cell for subsequent metabolism. This transportation is controlled by specific transporters encoded by genes such as MPH2, MPH3, AGT1, MTT1, MALx1, and other less-characterized genes [80,85]. Other factors include the copy number of transporter-encoding genes, variation in the promoter regions of these genes, and the positive regulators of Mal transporters. A crucial factor in this transport system is the polymorphism in key amino acids within the protein sequence of these transporters, as it has been reported that even a single change in the sequence can determine substrate preference and may even alter transporter activity entirely [80,86]. Furthermore, specific combinations of yeast species and sugars change the growth and fermentation characteristics of the yeast. In Kluyver-effect-positive yeast, such as Kluyveromyces lactis, certain sugars can only be used under respiratory conditions. For these yeast, respiratory inhibitors and respiratory-deficient mutations block all growth on maltose [82]. Alternatively, in Torulaspora delbrueckii, the consumption of maltose is repressed in the presence of glucose and induced in its absence [87].

4.2. Yeast Behavior as a Response to Oxygen

The Saccharomyces sensu stricto clade/complex includes S. cerevisiae, S. paradoxus, S. cariocanus, S. uvarum, S. mikatae, S. kudriavzevii, S. arboricola, and S. eubayanus. Some important natural hybrids found in brewing are S. pastorianus and S. bayanus [88]. Many yeasts in the Saccharomyces spp. are Crabtree-positive yeasts, able to convert sugar to ethanol and CO₂ under both aerobic and anaerobic conditions [89,90]. This effect is dependent on sugar composition and concentrations and S. cerevisiae switches to a mixed respiro-fermentative metabolism, producing ethanol at a glucose level of 0.8 mM or higher [91,92]. The sugar composition of the media and oxygen availability are thus the two key environmental drivers that impact the fate of carbon metabolism in yeast and thereby influence the ethanol yield and desirable volatile production in beer [89,92].

Crabtree-negative yeasts are ideal for NABLAB production due to their ability to switch between respiration and fermentation, potentially limiting ethanol production in the presence of oxygen. However, despite several studies on the Crabtree phenomenon, its regulation is not fully resolved across different species and conditions and requires more investigation. The short-term Crabtree effect is defined as the immediate triggering of aerobic alcoholic fermentation upon the sudden excess in glucose in a previously glucose-limited, respiratory culture. Once the glucose is depleted, ethanol metabolism through respiration takes place in a process called diauxic shift. The long-term Crabtree effect is defined as the sustained respiro-fermentative metabolism above a strain-specific sugar threshold [90,93]. Under aerobic conditions, the anaerobic pathway can also be activated when the rate of uptake of biochemical oxygen demand exceeds the rate of oxygen supply, indicated by the production of glycerol [89,94]. In contrast, in “Crabtree-negative yeast”, the fermentative pathway is carried out only when oxygen becomes limited. In this case, glucose concentration is not a factor in inhibiting respiration. This means that Crabtree-negative yeasts respire sugars under aerobic conditions regardless of sugar concentration, to yield CO₂ along with biomass and other non-ethanolic by-products [73]. Hence, the measurement of dissolved oxygen in the wort is important to better understand and control the metabolism of NSYs in order to reduce ethanol [95]. Hop iso-α-acids have a significant effect on the growth of several NSY strains manifesting either as prolonged lag times or the complete inhibition of growth [31]. Apart from the Crabtree effect, some other oxygen/sugar-linked phenotypes have been described in yeasts, such as the Custer effect, the Kluyver effect, and the Pasteur effect [96,97]. Contrary to the Crabtree effect, the Custer effect is known as the inhibition of alcoholic fermentation (and growth) upon an abrupt shift to anaerobic conditions in certain yeasts, for example Brettanomyces/Dekkera spp. [96,98,99].

4.3. Alternate Carbon Sinks in Yeast Metabolism in the Context of Barley Wort Composition

4.3.1. Glycerol Production

Under aerobic conditions, excess NADH produced during assimilation and metabolism can be shuttled to the mitochondria to be reoxidized via oxidative phosphorylation, helping meet ATP demands for growth and regenerating NAD⁺ for continued metabolism [100,101]. However, under anaerobic conditions, this respiratory reoxidation is not possible due to the absence of oxygen as the terminal electron acceptor [101]. Although alcoholic fermentation is itself a redox-neutral process reoxidizing NADH generated in glycolysis, net NADH during anaerobic fermentation can still accumulate, requiring further redox balancing [102]. This issue is usually solved in S. cerevisiae by reducing glucose to glycerol, which consumes NADH and helps maintain redox balance (Figure 2). Glycerol is formed by the reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate by the Gpd enzyme, followed by phosphate removal by Gpp to give glycerol. This process is largely performed during the early/growth phase of fermentation [102,103]. The S. cerevisiae genome encodes two isozymes of each of Gpd and Gpp that function differently in the response to either osmotic stress or redox rebalancing needs [104,105]. Interestingly, Gpd2 is primarily involved in redox balance, and the degree of induction of this isoenzyme varies according to different yeast types, which may drive differing glycerol levels during fermentation depending on the yeast strain used [106,107].

After ethanol, glycerol is most impactful metabolite produced by NSYs during fermentation [108]. It contributes to smoothness (mouthfeel), sweetness, and complexity, dependent on the substrate profile [38,51,104]. Typically, Metschnikowia, Hanseniaspora, and Torulaspora spp. are characterized by high glycerol production in beer [58]. Several factors come into play in influencing the production of glycerol, which is generally produced more by non-Saccharomyces yeasts than Saccharomyces spp. The sugar levels, ethanol production, and redox balance, as well as yeast species and strains, influence the production of glycerol [105,108]. Typically, in high-sugar environments such as in wine grapes, this glycerol production helps combat osmotic stress in NSYs [60]. Oxygen availability is also important, as NSYs produce more glycerol with the reduction of oxygen availability [38,109]. A study conducted by Gutiérrez in 2018 examined the glycerol production of different NSYs in wine, cider, and beer [110]. They found that the Kluyveromyces spp., Pichia kluyveri, and Saccharomycodes ludwigii consistently produced high amounts of glycerol across the different matrices. Overall, the ability of NSYs to produce higher glycerol is one of the main differentiating features from Saccharomyces spp. used in wine and beer, but these outcomes almost always vary according to strain differences and inoculation strategies (pure vs. co-inoculation) [105]. Benito et al. found that while in general, Schizosaccharomyces pombe strains showed the highest concentrations of glycerol, there were some that produced comparable amounts to S. cerevisiae [111]. Similarly, certain studies showed that wines produced by T. delbrueckii had significantly higher concentrations of glycerol in comparison to S. cerevisiae, while others showed that T. delbrueckii inoculated in a mixed fermentation with S. cerevisiae did not reflect this increase compared to pure S. cerevisiae ferments [112]. This might indicate that, along with fermentation parameters, yeast species and strains seem to behave differently [38]. Most of the studies measuring glycerol production are however related to wine making and assumed to have similar results in brewing [110].

4.3.2. Organic Acid Production

Organic acids in beer are typically either derived from the wort or formed during fermentation [113,114]. The total acid in traditional beers is typically low, going up to ~2 g/L depending on the style and production method [115]. Specific strains of NSYs can be used to target organic acids that affect beer flavor and acidity [116,117]. The main organic acid produced by yeast can be categorized as either volatile or non-volatile [114]. Acetic acid, the predominant compound in vinegar, can increase under fermentation conditions to help maintain the redox balance [118]. In the beer matrix, it typically has a sensory threshold of 175 mg/L [119,120]. The non-volatile acids produced by yeasts in beer are oxalic acid, citric acid, malic acid, fumaric acid, succinic acid, lactic acid, and pyruvic acid [114]. These acids are linked to central carbon metabolism (glycolysis, the citric acid cycle, amino acids, and fatty acid metabolism), and the proportion of each acid is controlled by both the yeast species as well as the strain. Lachancea spp. (especially L. thermotolerans) have shown to produce a distinctly high amount of lactic acid (Table 1). This type of NSY is ideal for the production of sour beers instead of relying on lactic acid bacteria that are prone to causing a high amount of contamination in breweries [116]. Moreover, the bio-acidification of the NABLAB produced helps in the shelf life and stability of the product, by making it more resistant to microbes [121]. These metabolites are produced due to the heterolactic fermentation of sugar into lactic acid, ethanol, and CO₂, along with pleasant aromatic and flavor compounds. It can also be produced by the transformation of malic acid produced due to the TCA cycle [122]. In wine fermentation, these yeasts have been reported to increase the levels of ethyl lactate, a fruity ester that is also found in high levels in some Belgian beers like Lambic and Gueuze [123,124]. A study conducted by Pirrone et al. 2025 displayed the effectiveness of five different Lachancea thermotolerans strains in the production of sour beers with lactic acid contents in a 0.33-to-0.45 g/L range, leading to a pH between 3.0 and 3.9 [125]. Lactic acid is softer and milder compared to tartaric and malic acid found in wine, providing a creamier mouthfeel to wine, though levels may vary widely depending on the process.

Another NSY linked with distinct organic acid production is Metschnikowia spp. In wine, it is reported to reduce malic acid but increases in fumaric acid have also been observed [126]. Under anaerobic conditions, a decrease in ethanol by Metschnikowia has been associated with increases in the production of alternative carbon sinks like organic acids and glycerol [126,127,128]. Fumarate is typically not a dominant organic acid produced during fermentation; however, it can be elevated in fermentations with M. pulcherrima [124]. In the branched TCA cycle, fumaric acid is produced from malate via fumarase activity in the reductive branch of the cycle. This fumarate is then converted to succinate by fumarate reductase. Fumaric acid specifically produces a sharp, long-lasting, astringent sourness detected at concentrations above 0.6 g/L in wine [129]. However, there have only been a few studies showing fumaric acid in beer in negligible amounts, so evidence of elevation in fumarate due to Metschnikowia spp. in NABLABs still faces a key research gap [130]. Succinate also increases along with fumarate in the sequential fermentation of Metschnikowia pulcherrima and Saccharomyces cerevisiae in wine. While the cause of this increase is still unclear, this is a display of the rerouting of carbon away from ethanol production [127]. Succinic acid may be formed via TCA-linked pathways and the decomposition of amino acids during fermentation [131,132]. Some studies have shown an increase in succinic acid in fruit spirits produced by both Torulaspora delbrueckii as well as Lachancea thermotolerans [133]. Succinic acid is usually produced in wine from 200 mg/L to 2 g/L and produces a distinct salty, sour, and bitter taste in fermented beverages. It also has the potential to react with other molecules to form esters such as ethyl and diethyl succinate, having a positive sensory impact [132].

5. Wort Composition Effects on NSY Fermentation

5.1. Role of Fermentable Sugars in Wort in NABLAB Production

Malted barley has been used by brewers for centuries to produce various styles of beer. After malting, mashing and fermentation are crucial steps that determine the style and quality of a brew. In the mashing stage, milled malt is mixed with water and heated to gelatinize starch and activate endogenous enzymes such as α- and β-amylases [25,134]. This step, known as saccharification, controls the concentration and spectrum of fermentable sugars that will then be used by the yeast to drive fermentation and the ethanol concentration of the final beer [135].

The fermentable sugar profile of a typical pale ale barley malt consists of ~70% maltose, ~14% glucose, and ~15% maltotriose, with lower levels of fructose and sucrose [48,136]. This sugar profile is a result of the activity of α-amylase, β-amylase, and limit dextrinase on gelatinized starch granules during mashing. The α-amylase cleaves starch randomly into dextrins of varying sizes, with limit dextrinase removing branches and β-amylase acting on non-reducing ends to release maltose. The temperature for barley starch gelatinization (~62–65 °C) is within the optimum temperature range for these enzymes [137]. This temperature is further suitable for the activity of the heat-sensitive β-amylase, allowing a single temperature infusion mash to be used for the efficient production of maltose, acting as the majority fermentable sugar [138]. Once the wort is produced from the mash, it can serve as a substrate rich in fermentable sugars that the yeast can utilize to anaerobically produce ethanol and carbon dioxide.

5.2. Mashing Strategies for Modifying Fermentable Sugar Profile of Wort

The mashing step in brewing is crucial in the production of fermentable sugars and dextrins from starch in traditional brewing and even more so in NABLAB brewing. As mentioned in the previous section, the fermentable sugar profile in a typical barley malt is dominated by maltose and maltotriose that NSYs cannot utilize due to a lack of specific transporters [28]. Since the β-amylase is heat-sensitive and has a small temperature window, multiple NABLAB approaches intentionally deactivate β-amylase to produce a low-maltose wort [7,10,130]. One strategy to do this is by mashing barley at a high temperature of 75–80 °C to maintain α-amylase, which breaks down starch into unfermentable dextrins while deactivating β-amylase [5,7,137]. The use of a higher liquor-to-grist ratio further helps in modulating the total fermentable sugar produced. For NABLAB production, many studies have targeted a starting gravity of 6–7 °P, which is lower than the starting gravity of traditional alcoholic beers (12°Plato) [48,135,137,139]. Other pathways to change the wort sugar profile could be utilizing the spent grain from a primary mash to control sugar production or using a barley malt variety that is deficient in β-amylase [12]. However, lower-fermentability/low-gravity worts can also limit the growth of NSYs and flavor development, contributing to underdeveloped flavor profiles [9,29,78,140].

Another approach to changing wort profiles is the addition of exogenous starch-degrading enzymes like amyloglucosidase to increase the relative concentrations of fermentable glucose by decreasing concentrations of maltose in barley malt while [141,142]. A recent study found that adding exogenous amyloglucosidase to low-gravity worts at a specific dose and incubation time helps improve the fermentation performance of maltose- and maltotriose-negative NSYs due to the presence of higher levels of fermentable sugars, leading to lower ethanol production compared to Saccharomyces yeast for the same initial wort conditions [130]. They found that the Crabtree-negative NSYs are able to metabolize the glucose to grow under microaerobic conditions and also divert the carbon towards sinks other than ethanol production such as glycerol, organic acids, and biomass, expanding on findings from previous studies [73,127]. Thus, mashing alterations can be a key tool for brewers to control final alcohol levels.

5.3. Free Amino Nitrogen and Yeast Nutrition in Low Gravity Wort

It is important to examine the influence of free amino nitrogen to understand the yeast metabolism relating to the production of aroma-active compounds such as higher ethanol [10,65,84]. The yeast requires ~200–250 mg/L of free amino nitrogen (FAN). Modern barley malts usually have sufficient FAN to support these nutrition requirements [143].

For active fermentation, especially at low gravity, care should be taken in the measurement of FAN. Studies report a lowering of FAN to 55–99 mg/L at 7°Plato, usually considered too low for yeast performance. However, the FAN requirements are lower in low-gravity worts due to fewer fermentable sugars, leading to less yeast growth and metabolic activity [10]. For this reason, there should be a balance between the FAN and fermentable sugars in the wort.

5.4. Effect of Dissolved Oxygen in Wort

Initial oxygenation is crucial for healthy yeast growth and fermentation performance and insufficient oxygen can contribute stress-associated flavor issues [144,145]. The target range of dissolved oxygen (DO) in brewing is typically 7–12 mg/L (ppm) before pitching; therefore, a DO meter should be utilized to confirm the oxygen content of the wort [146,147]. However, aeration can have a major effect on the carbohydrate utilization of Kluyver-effect-positive NSYs, resulting in their ability to utilize sugars such as maltose that they cannot use anaerobically [82]. While theoretically this could mean that a decrease in residual sugars without increasing ethanol could be achieved, the net effects on both ethanol and sensory properties of increasing DO with NSY fermentations requires additional investigation.

5.5. Fermentation Parameters

Other parameters relating to the fermentation kinetics of the yeast are important considerations for reaching the target ABV%. Different yeast species and strains have different fermentation rates given the same starting conditions; as a result, there is a need to tailor the fermentation period according to the yeast species used and the amounts of residual sugars that can be tolerated in the final product [21,24,58,148]. Factors such as the temperature, aeration, pH, sugar profile, and pitching rate also need to be taken into account while designing ferments using different NSYs [13]. Apart from the yeast strain, the temperature is the most noticeable factor in producing fruit alcohols and esters. It has a critical effect on the expression of a gene responsible for encoding a permease that allows the transport of amino acids inside the yeast cells, thus affecting the production of higher alcohol [61,149]. A higher temperature of fermentation increases esters, higher alcohols, and ethanol [146]. A temperature range of 15–20 °C is typical for fermentation to produce NABLABs, with preference given to lower temperatures to slow down NSY metabolic activity [12,19,58,150,151]. The pH of wort is typically between 5 and 5.5 in traditional brewing, but in NABLAB production, a lower pH (4.9) is preferred as an added hurdle to ensure microbial stability during fermentation in the absence of ethanol [57,136,152]. Organic acid production can further reduce the pH and help in preventing spoilage [153]. The final pH of the NABLAB should be less than 4.3 for product stability [57]

A typical pitch rate in NABLAB production using NSYs is usually 10⁶ to 10⁷ cells/mL. The pitch rate can also influence the fermentation and flavor production depending on the NSY, showing a positive correlation with ester formation [154]. Ultimately, the fermentation parameters should be selected with the goal of producing the least amount of ethanol along with reducing excessive sweetness and worty off-flavors while also producing significant desirable flavor compounds [6,39,48].

6. Sensory Outcomes and Flavor Balance

6.1. Sweetness Drivers and Mitigation Strategies

A taste parameter often associated with NABLABs is sweetness. NABLAB production with high maltose, when fermented by maltose-negative yeast, often leaves behind high amounts of unfermented residual sugars [155,156]. Several studies report that NABLAB products from limited or inhibited fermentation have immature and excessively sweet flavors and perceived mouthfeel due to underdeveloped flavor compounds [20,22,157]. Lafontaine et al. in 2020 [33], analysed the chemical and sensory profiles of 42 different non-alcoholic beer brands/styles available globally and produced through different brewing techniques. They concluded that sweet NABs with citrus, tropical or stone fruit, aromas were preferred by consumers. Consumers also gave positive ratings to lager and wheat-style NABs that were sweet, with honey-like aromas [33]. This perhaps indicates that sweetness is deemed acceptable in NABLABs when it is accompanied by other tastes/aromas that consumers typically associate with sweet foods. Another study conducted by Schmelzle et al. in 2013 showed that while beers with a degree of sweetness and fruity characteristics are preferred by consumers, NABLABs made from inhibited or suppressed fermentation had a sweetness assessment of being “much too strong” or “too strong” [34]. Brewers are therefore advised to avoid excessive malty, honey-like, or bread-like notes linked to unfermented residual sugars and Strecker aldehydes in NABLAB produced by maltose-negative NSYs [8,29,156]. The harmony or balance between sweetness, palate fullness, bitterness, and acidity is an important aspect for NABLABs rather than the individual notes themselves. There is a positive correlation between the sweetness and thickness/body of NABLABs, as both are controlled by regulating the initial fermentable sugar profile and gravity. Due to this correlation between sweetness and body, glycerol produced by NSYs in high amounts also plays a part in contributing to the overall sensory properties in NABLABs [158].

The absolute sweetness intensity of the five natural sugars is as follows: fructose > sucrose > glucose > maltose > lactose [159,160,161]; this suggests that it is not just total sugar, but the profile of sugars in the final beer that is important. Additionally, characteristics like bitterness, carbonation, and acidity can also help in masking sweetness [162]. Bitterness, carbonation, and acidity in NABLABs can be controlled by the hopping regime, controlling carbonation, and yeast-driven/exogenous acidification. In NSYs like Lachancea thermotolerans, the higher amount of lactic acid produces a sour taste that can potentially help in producing sour beer varieties with less malty or unharmonious sweet tastes [163,164]. “Worty” notes typically occur due to the short fermentation period of a NABLAB, not giving the NSY time to decrease aldehyde to less flavor-active compounds. Aldehydes like 2-methylbutanal, 3-methylbutanal, and methional have an extremely low threshold of perception. Glycerol produced by NSYs is able to “retain” undesirable aldehydes in NABLABs and hence has potential to reduce their perception in NSY-fermented NABLABs [165].

6.2. Aroma- and Flavor-Active Compounds from Non-Saccharomyces Yeast

NSY explored in NABLAB production can be sourced from many fermented foods and beverages, with the most common source being wine-associated yeast [166]. Additional NSYs have also been isolated from other fermentations (e.g., kombucha, sourdough, dairy) and non-traditional starters like daqu, chicha, and loquat [167,168,169,170]. NSYs such as Pichia kluyveri, Hanseniaspora uvarum, and Torulaspora delbrueckii are known to produce complex aromas in the form of esters such as isoamyl acetate, ethyl hexanoate, and ethyl acetate, and higher alcohols such as 2-phenylethanol [151]. This characteristic makes NSYs appealing in the production of NABLABs with unique and complex flavor profiles. However, the ability to produce desirable aroma compounds varies substantially between species, creating a need for systematic screening [171]. These characteristics are often overshadowed by the high amount of residual maltose that NSYs are unable to utilize. Apart from this, some NSYs can contribute to biotransformation by producing enzymes such as β-glucosidase and β-lyase, which can release aroma-active terpenes and polyfunctional thiols from hop-derived precursors. The β-glucosidase activity of NSYs is much lower in a wort matrix compared to grape must due to differences in pH, proteins, and precursor pools. This is supported by evidence of the increased release of varietal thiols such as 3-Methylpyridine (3MP) and 3-mercaptohexan-1-ol (3MH) relative to S. cerevisiae in wine [42,52]. In beer, there have been limited studies that have reported an increase in 3MH with β -lyase activity, depending on the strain used. Moreover, the resulting increase in 3MH is to similar levels as S. cerevisiae. This biotransformation is limited to Torulospora and Saccharomyces spp. and is rare in other yeast species. Similarly, Sharp et al. reported that even when yeasts show higher β-glucosidase activity, their effect on glycosidically bound terpenes depends on the hopping regime and cultivar [42,149]. Indeed, a study conducted by A. King in 2000 showed that yeast can interconvert monoterpenoids, including the reduction of geraniol to citronellol, as well as the conversion of geraniol and nerol to linalool [172].

7. Conclusions

Commercial NABLAB production often combines a variety of biological and physical methods to obtain the desired alcohol levels and sensory quality. The available literature related to biological methods of NABLAB production highlights the importance of non-Saccharomyces yeasts in brewing. Among maltose- and maltotriose-negative yeasts, fermentation performance is species- and strain-dependent. To better control the production of ethanol through biological methods, it is therefore crucial to understand the role of wort composition and its interaction with different yeasts. While ethanol production is typically the major pathway, alternative fates of carbon include glycerol, organic acids, biomass, indirect aroma-active volatile compounds, and CO₂ production. These minor pathways can lead to meaningful differences in mouthfeel, sweetness perception, acidity, and aroma complexity.

Current strategies to improve ethanol control and sensory balance include tailoring the wort profile to the metabolic capabilities of the selected NSY strains (e.g., using a low gravity or low pH wort, lowering maltose/maltotriose concentrations with high mashing temperatures to deactivate β-amylase, and modulating the wort sugar profile with exogenous amylase enzymes). A major gap remains in examining the effects of wort oxygenation on the NSY metabolism and NABLAB production as some NSYs exhibit Crabtree-negative and Kluyver-effect-positive characteristics, which may have a complex relationship with sensory properties, ethanol production, and residual sugar levels. Additionally, the introduction of mixed cultures in brewing may further help in diversifying the NABLAB flavor portfolio as in winemaking, based on interspecies interactions in wort media. However, the growing body of knowledge on the use of NSYs in brewing has opened many exciting opportunities for NABLAB production.