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Article

Functional Non-Alcoholic Beer Fermented with Potential Probiotic Yeasts

by
Peter Vaštík
1,
Ján Brunner
1,
Rudolf Jung
2,
Tatiana Klempová
1,
Katarína Furdíková
1,
Daniela Šmogrovičová
1 and
Pavel Dostálek
2,*
1
Institute of Biotechnology, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 812 37 Bratislava, Slovakia
2
Department of Biotechnology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Technická 5, 166 28 Prague, Czech Republic
*
Author to whom correspondence should be addressed.
Beverages 2025, 11(5), 140; https://doi.org/10.3390/beverages11050140
Submission received: 8 August 2025 / Revised: 29 August 2025 / Accepted: 18 September 2025 / Published: 22 September 2025
(This article belongs to the Section Malting, Brewing and Beer)
Browse Figures
Versions Notes

Abstract

The development of non-alcoholic beer (NAB) with health benefits, using non-conventional potential probiotic yeasts, offers an interesting alternative to standard NAB brewing strategies. In this study, potential probiotic non-Saccharomyces yeasts Pichia manshurica, Kluyveromyces lactis, and Kluyveromyces marxianus, along with commercial probiotic yeast Saccharomyces boulardii, were characterised and tested for functional NAB production, whereas P. manshurica was used in NAB production for the first time. Growth and viability were assessed across a range of temperatures, pH, and iso-α-bitter acids. The tested yeasts withstood conditions typical of the beer matrix and human digestive tract and had a positive phenolic off-flavour phenotype. Two strains, K. lactis and K. marxianus, showed strong β-glucosidase activity, which may enhance beverage aroma complexity. Ethanol levels in beers fermented with non-Saccharomyces yeasts remained below the NAB limit (≤0.5% v/v). An analysis of volatile organic compound profiles revealed the potential of these yeasts to produce higher alcohols and esters valuable from a brewer’s perspective. This study provides valuable insight into novel probiotic fermentations and the potential application of unconventional yeasts in functional, aromatic, and health-oriented non-alcoholic beverages.

Graphical Abstract

1. Introduction

The traditional beer markets are thriving, and brewers aim to meet emerging consumer trends [1]. Although the non-alcoholic beer (NAB) market was valued at USD 36.7 billion, it represents only 4.3% of global beer production [2]. Rising interest in healthier foods has led to functional beers, such as probiotic or gluten-free options [3]. The probiotic market, worth USD 61.2 billion in 2021, is projected to grow by 7.7% by 2030 [4]. Beverages as functional matrices are becoming more common, yet adding probiotics to beer is challenging due to its harsh environment [5]. Consumers increasingly prefer beers with lowered ethanol content aligned with a healthier lifestyle [6], while breweries preserve capital from lower tax burdens [7]. According to EU Regulation 1169/2011, beverages over 1.2% vol. are alcoholic, but most countries define NAB as ≤0.5% vol [8]. NAB can be made by limiting ethanol during fermentation or removing it post-production, but this can cause off-flavours and aroma loss [9,10]. The use of non-maltose fermenting yeasts helped improve NAB sensory qualities [10,11,12].
Probiotics are live microorganisms with health benefits when consumed in sufficient amounts [13]. Considering the potential adverse health effects of ethanol, functional probiotic beers are most relevant in the form of non-alcoholic beers [14]. Labels may state colony forming unit (CFU) levels, but high counts do not always mean better health effects [15]. Probiotics can colonise the gut, compete with pathogens, and produce beneficial compounds [16]. Though only few probiotic yeasts (PYs), like Saccharomyces boulardii or Kluyveromyces fragilis B0399, are used, other genera also show probiotic potential, including tolerance to low pH, bile salts, and antimicrobial activity [17].
S. boulardii, a generally recognized as safe (GRAS) organism [18], is a subtype of S. cerevisiae and produces ethanol, CO2, and bioactives like γ-aminobutyric acid (GABA) and B vitamins [19]. K. lactis, studied since the 1960s [20], ferments lactose using LAC12 and LAC4 genes [21] and can produce ethanol even anaerobically [22]. K. marxianus, a thermotolerant, Crabtree-negative yeast, survives at 52 °C and can produce ethanol above 40 °C, but its inability to ferment maltose makes it suitable for NAB [23]. Pichia manshurica, found in fermented foods and wines, is associated with biofilm and volatile phenol production [24]. Though never used in beer, it showed survival potential under stress factors [25] and successfully enhanced the vinegar aroma profile in one study [26].
This study applies S. boulardii, K. lactis, K. marxianus, and P. manshurica as sole fermentation cultures in functional NAB production. Results support the concept of using probiotic yeasts to develop next-generation health-promoting beverages.

2. Materials and Methods

2.1. Microorganisms

The yeast strains employed in this study (Table 1) were cultivated on yeast, peptone, dextrose, agar (YPDA) medium [10 g·L−1 yeast extract (Oxoid, ThermoFisher Scientific, Waltham, MA, USA), 10 g·L−1 peptone (Thermo Scientific™, Waltham, MA, USA), 20 g·L−1 glucose (Merck, Darmstadt, Germany), and g·L−1 agar (Carl Roth, GmbH, Karlsruhe, Germany); pH 6.2] and preserved at 4 °C.

2.2. Preparation of Yeast Inoculum

Yeast inocula used in the experiments were obtained through a 24 h submerged cultivation of individual yeast strains in liquid yeast, peptone, dextrose (YPD) medium [10 g·L−1 yeast extract, 20 g·L−1 glucose, 10 g·L−1 peptone (Thermo Scientific™, Waltham, MA, USA); pH 6.2; 20 mL in 100 mL Erlenmeyer flasks] using an orbital shaker (Biosan ES-20, Riga, Latvia) operated at 2 Hz and maintained at 28 °C.

2.3. Saccharide Fermentation

The ability of yeast strains to ferment specific saccharides (glucose, maltose, lactose) was assessed as described previously [27], using glass tubes equipped with inverted Durham tubes. The release of CO2, indicative of saccharide fermentation, was evaluated following a 7-day static cultivation at 25 °C. All experiments were conducted in triplicate.

2.4. β-Glucosidase Activity

Yeast strains with positive β-glucosidase activity are capable of hydrolysing aesculin as the sole carbon source to glucose and aesculetin, which reacts with the present iron ions to form a dark compound. Tested yeast strains were inoculated onto plates with medium containing 1.0 g·L−1 of aesculin (Fisher Scientific, Waltham, MA, USA), 0.5 g·L−1 of iron (III) citrate 3-hydrate (Acros Organics®, Thermo Scientific™, Waltham, MA, USA), 8.0 g·L−1 of yeast extract, 15 g·L−1 of agar, and they were incubated for 24 h at 25 °C. The intensity of β-glucosidase activity was evaluated based on the formation of the dark zone and the intensity of diffusate colouring. Experiments were performed in triplicate.

2.5. Phenolic Off-Flavour (POF) Phenotype

Yeasts capable of decarboxylating ferulic acid into the formation of 4-vinyl guaiacol (4-VG), which imparts beer with a clove-like aroma, can be characterised by their positive (POF+) or negative (POF−) phenotype. Yeast starters used in experiments were prepared by a 24 h submerse cultivation of individual yeast strains in liquid YPD medium. For each yeast strain, 20 mL of pure and sterile YPD medium was poured into a glass tube with an addition of 0.2 mL of 1% (v/v) ferulic acid solution prepared by adding ferulic acid (Merck, Darmstadt, Germany) into 96% (v/v) ethanol (CentralChem®, Bratislava, Slovakia). Glass tubes were inoculated by the tested yeast strains in triplicate. Tubes were then sealed and statically incubated at 25 °C for 24 h. Evaluation was carried out by six people and performed by a sensorial analysis comparison of glass tubes containing the tested yeast strains against controls, where as a positive control (POF+) yeast SafBrew™ LA-01 was used. As a negative control (POF−), yeast LalBrew® LoNa™ was used.

2.6. Tolerances of Different Conditions

To determine the sensitivity of strains to different temperature conditions, 1 × 106 Cells·mL−1 of liquid yeast starters were cultivated at 4 °C, 20 °C, and 37 °C for 24 h in sterile glass tubes each containing YPD medium.
The sensitivity of strains to different pH conditions (3, 4, 5, and 6) was evaluated by cultivating 1 × 106 Cells·mL−1 of fresh liquid yeast starter at 37 °C for 24 h in sterile glass tubes each containing YPD medium, where the pH was adjusted by adding 35% (v/v) of HCl (CentralChem®, Bratislava, Slovakia). The sensitivity of strains to different concentrations of iso-α-bitter acids in terms of IBU (international bitterness unit) (0, 10, 30, and 50) was evaluated by cultivating 1 × 106 Cells·mL−1 of fresh liquid yeast starter at 25 °C for 24 h in sterile glass tubes each containing YPD medium, where IBUs were adjusted by the addition of iso-α-bitter acid solution (Brewferm®, Richmond, UK).
The tolerance at different temperatures, pH, concentrations, and iso-α-bitter acids was evaluated based on the growth of the yeast culture, which was determined by measuring the optical density of the biomass suspension at a wavelength of 600 nm (ΔOD600 nm) against pure YPD medium used as a blank. The viability of yeast cells was determined microscopically using staining with 0.1% (w/w) Methylene Blue solution (Merck, Darmstadt, Germany). Experiments were performed in triplicate.

2.7. Fermentation and Maturation

For beer preparation, 480 mL of wort (8 °P made from Pilsen malt and Žatecký poloraný červenák hops (Saaz)) prepared at 25 L Laboratory Microbrewery (Braumeister, Speidel, Germany) in 500 mL fermentation PET flasks was inoculated with yeast starters with a pitch rate of 1 × 106 Cells·mL−1. Flasks were closed and fermentation proceeded at 20 °C for 2 days after which maturation proceeded at 3 °C for 3 weeks. Beers were then stabilised by a pascalisation procedure at 400 MPa for 3 min. Finally, fermented beer samples were analysed for the composition of residual saccharides, organic acids, glycerol, and ethanol, as well as for a profile of the main volatile organic compounds (VOCs). The viability of cell cultures in the final beer samples was determined after the stabilisation procedure. Fermentation experiments were performed in triplicate.

2.8. Beer Analyses

2.8.1. Basic Beer Parameters

The ethanol concentration and pH of beer and wort samples were determined with a DMA 4500 M density metre (Anton Paar, GmbH, Graz, Austria) coupled with the Alcolyzer Beer ME, Haze QC ME Turbidity Module, and pH ME Beverage Module. Before analysis, fermented beer samples were centrifuged using Rotina 420 (Hettich, Bäch, Switzerland) (10 min, 2524× g), degassed in VGT 1730T (GT SONIC, Shenzhen, China) by 30 min ultrasonication, and analysed in triplicate.

2.8.2. Organic Compound Analysis by HPLC-RID-DAD

The beer and wort samples were centrifuged (10 min, 2511× g), and the supernatant was diluted with deionized water if needed. An Agilent 1260 HPLC system (Santa Clara, CA, USA) coupled to an RI (refractive index) and DAD (diode array detector) using an Aminex HPX-87H column (300 mm, 7.8 mm; Bio-Rad Laboratories, Hercules, CA, USA) was used for HPLC analysis. Sulfuric acid (5 mmol·L−1) served as the mobile phase, applied at a flow rate of 0.6 mL·min−1. The separation of analytes was performed at 25 °C, where the injection volume of the sample was 20 μL. The signal was detected by RIDs and DADs. Precise concentrations of glucose, maltose, glycerol, and acetic, lactic, and citric acids were quantified using the single standard addition method. Standards with a purity of ≥99.5% were obtained from Merck (Darmstadt, Germany). Beer and wort samples were analysed in triplicate.

2.8.3. Volatile Organic Compound Analysis by HS-SPME-GC-MS

The beer samples were cooled and stored at 4 °C. An aliquot of 50 mL from each beer sample was centrifuged (10 °C, 5054× g, 10 min), and the supernatant was transferred into a 50 mL flask and sealed. The flasks were shaken for 3 min to remove dissolved CO2. Subsequently, 2 g of NaCl (≥99.9% purity, Pentachemicals, Prague, Czech Republic) were added to a 20 mL dark vial containing 10 mL of beer sample and 100 µL of the internal standard (IS) solution, which consisted of ethyl heptanoate (≥99% purity, Sigma Aldrich, Taufkirchen, Germany) and 3-octanol (≥99% purity, Sigma Aldrich, Saint Louis, MO, USA). The vial was vortexed for 30 s to dissolve NaCl and homogenise the sample. Volatile organic compounds (VOCs) were identified and quantified according to the method described in [27]. All beer samples were analysed in triplicate.

3. Results and Discussion

3.1. Yeast Characterisation

Fermentation tests showed that all strains were able to ferment glucose (Table 2). Unlike the probiotic strain S. boulardii, the other four non-Saccharomyces yeast strains of the Kluyveromyces and Pichia genus were unable to ferment maltose—the most abundant saccharide in wort (Table 2)—making them proper candidates for non-alcoholic beer (NAB) production by the strategy of using maltose-negative yeast strains [28]. Potential probiotic strains K. lactis and K. marxianus were able to ferment lactose, as was previously confirmed in [29]. S. boulardii and both yeast strains of P. manshurica have not fermented lactose (Table 2). The determination of the β-glucosidase activity of tested yeasts proved that both strains of P. manshurica, as well as S. boulardii, had weak/delayed β-glucosidase activity after 24 h, whereas K. lactis and K. marxianus showed strong β-glucosidase activity (Table 2). Several studies have reported that yeasts with increased β-glucosidase activity play an important role in releasing aromatic aglycones from hops during fermentation [30], thus enhancing the aroma complexity of the final beverage. The positive phenolic off-flavour (POF+) phenotype was sensorially evaluated by the production of a clove-like aroma (4-vinyl guaiacol (4-VG)) by all tested yeasts (Table 2). Besides diacetyl and sulphur compounds, 4-VG is a mostly unwanted compound during beer production with the exception of few beer styles, e.g., German Hefeweizen and Belgian Wit beers, where 4-VG is considered a part of the aromatic profile [31]. Even though the tested yeasts were POF+, they might serve as a fermentation starter culture for brewing specific non-alcoholic wheat beers with a clove-like aroma.

3.2. Tolerance at Different Temperatures

Yeast growth at different temperatures represented as ΔOD600 nm values showed that the potential probiotic yeasts Kluyveromyces were unable to grow at 4 °C (Figure 1); however, the highest growth represented as a ΔOD600 nm value was observed in a medium incubated at 37 °C (Figure 1). According to [32], the yeast K. marxianus can withstand 45 °C, but as the human body temperature equals 37 °C, it was not our goal to test growth at ≥37 °C. According to [33], the optimal growth temperature for the probiotic yeast S. boulardii is 37 °C. This was supported by our results, and S. boulardii was able to grow sufficiently at a whole range of temperatures (Figure 1). The maturation of beer is performed at temperatures close to 0 °C. The growth of S. boulardii at low temperatures (4 °C) (Figure 1) might potentially lead to the unexpected fermentation of wort saccharides such as maltose or glucose due to the positive fermentability of these saccharides (Table 2); hence, the ethanol limit for NAB production should be maintained. The growth of both P. manshurica, represented as ΔOD600 nm values, was observed at 37 °C (Figure 1), whereas no growth was observed at 4 °C and growth was minimal at 20 °C. Results supported the suitability of the tested yeast to survive the human body’s temperature. Overall, the highest viability (Table 3) was determined when cultivating yeast at 37 °C, supporting the probability of survival of the tested yeasts in a human gastrointestinal tract. As for 20 °C, which is a temperature within the interval used for brewing ale-style beers, the viability for both Kluyveromyces and both Pichia yeast strains has decreased for more than 10% except for S. boulardii, which was the most viable yeast strain (97%).

3.3. pH Tolerance

The determination of pH tolerance was used to identify the growth behaviour of the studied yeasts to withstand the harsh conditions of the human stomach (pH 3), the fermentation medium of beer (pH 4–5), and the fermentation medium of wort (pH 6). Yeast growth for Pichia and Kluyveromyces sp. was strongly inhibited when exposed to a highly acidic environment close to the pH of the human stomach (pH = 3); only S. boulardii yeast was able to withstand this acidic environment and with the highest determined ΔOD600 nm values (Figure 2). According to [34], the probiotic yeast S. boulardii can survive under stomach conditions (pH 3), which was confirmed by our results. As for the other four non-Saccharomyces yeasts of Pichia and Kluyveromyces sp., an increased cell count (above the initial inoculated pitch rate of 106 Cells·mL−1) in the ΔOD600 nm values was detected at pH ≥ 3; cell count generally continued to increase up to pH 6. The viability of a traditional brewery yeast should normally lie in the range of 90–99% [35], and it is generally accepted that the live cell content of the yeast slurry used for subsequent fermentation should contain >95% of live cells, whereas high yeast viability allows for the production of high-quality beer [36]. However, our viability results revealed that Kluyveromyces and Pichia yeasts were not as viable as S. boulardii (69%) in acidic media (pH = 3) after 24 h at 37 °C due to their low percentage of viability, which was under 21% (Table 4). Different results were obtained when several K. marxianus strains were investigated in [37] in a harsh acidic environment (pH < 3). The results showed an initial reduction in cell count from 109 to 107 Cells·mL−1. Afterward, the results showed the growth of K. marxianus strains during the incubation period of 96 h. Stable intracellular pH is crucial for yeast growth and metabolic activity, as enzymatic functions depend on an intracellular pH environment, whereas significant deviations in extracellular pH can disrupt this balance, impairing enzyme activity and cellular function [38].

3.4. Tolerance to Iso-α-Bitter Acids

Hops are traditionally added during beer brewing as a bittering and flavouring agent [39]. Hops contain α-bitter acids which isomerize during the boiling step to form iso-α-bitter acids—compounds responsible for the bitterness of beer [40]. These substances report antimicrobial properties and protect the beer against the most common spoilage bacteria [41]. However, ref. [42] reported that iso-α-bitter acids can affect not only the growth of lactic acid bacteria but can also inhibit the growth of yeast S. cerevisiae at a concentration of iso-α-bitter acids above 500 mg·L−1 (equal to 500 International Bitterness Units—IBUs), which is approximately ten times higher than the concentrations required to inhibit bacterial growth. According to [14], most alcohol-free beers do not tend to exceed 30 IBUs. The growth of the tested yeasts was probed in the presence of 10, 30 and 50 IBUs (values characteristic for most of the beer styles), where no exceptional effects of different concentrations of iso-α-bitter acids in terms of different IBUs on yeast growth was detected (Figure 3). The viability of all strains in tested media with different IBUs remained above 95%.

3.5. Basic Beer Parameter Analysis

An important analytical parameter of beer is the ethanol concentration, which influences the sensory properties of beer, especially the fullness of flavour, but also the colloidal and biological stability of the beer [3]. The ethanol concentration in the produced beers ranged from 0.04 ± 0.01% (v/v) (PM1) to 1.52 ± 0.06% (v/v) (SBL) (Table 5). In all the analysed beers, except the beer produced with Saccharomyces boulardii, none displayed an ethanol concentration ≥0.5% (v/v), which is the limit concentration in alcohol-free beers [43]. The authors [44] also studied the use of the probiotic yeast S. boulardii and the influence of hops on its propagation and fermentation performance, and they produced beers with 4.67–3.26% (v/v) of ethanol. An important note should be considered as the term “probiotic beer” (containing 1 × 109 CFU, beneficial for health) [15] might be in conflict with the term “alcoholic beer” (generally containing more than 1.2% v/v of ethanol), where ethanol is considered a drug. In the characterisation of beers, pH is an important quality indicator which influences the foaminess, clarity, and microbiological and colloidal stability of the beer [45]. During the fermentation of wort, organic acids are produced by yeast, which leads to a decrease in the pH value of the product. The average pH of common beers ranges from 4.3 to 4.7 [46], while the pH of the produced beers ranged from 4.83 ± 0.02 (SBL) to 5.80 ± 0.01 (PM1) (Table 5), which was supposedly due to the short fermentation times (2 days). The viability of the tested yeasts with methylene blue staining showed that the pascalisation procedure successfully inactivated the yeasts in all beer samples fermented with tested yeasts (Table 1) to prevent further fermentation activity.

3.6. Organic Compound HPLC Analysis

The saccharide composition of beer can greatly affect the resulting taste of beer. Saccharides such as maltose strongly contribute to the body of the beer, while glucose and sucrose contribute to the sweet taste of the beer [47]. In comparison with the used wort, a minimal decrease in glucose and maltose were observed in beers fermented with Kluyveromyces and Pichia yeasts (Table 6). It is known that many yeasts can assimilate certain mono- or oligosaccharides aerobically but not anaerobically (fermentation), and this phenomenon is known as the Kluyver effect [48]. Oxygen availability plays a key role in determining the fermentation pattern of K. lactis. As oxygen availability decreases, overall glucose metabolism slows down, resulting in reduced fermentation activity [49]. This might be one of the answers to the results shown in Table 6, where only a small proportion of glucose and maltose was consumed by the non-Saccharomyces yeasts K. lactis, K. marxianus, and both strains of P. manshurica. In comparison, beer fermented with the probiotic yeast S. boulardii (SBL) had no residual glucose, which was presumably utilised during the first 2 days of beer fermentation (Table 6). This is supported by the obtained results from saccharide fermentation tests, where after 24 h, glucose was already being fermented (Table 2). As for maltose, the most abundant saccharide present in the beer wort [39], results showed that its final concentration decreased strongly during fermentation from 36.8 ± 0.5 g·L−1 (wort 8 °P) to 24.1 ± 0.3 g·L−1 in the beer fermented by S. boulardii (SBL), as was expected (maltose-positive strain) (Table 2), and ethanol (1.52 ± 0.06% (v/v)) was produced. Glycerol concentrations were below the perception threshold level, which in beer is 10 g·L−1 [39]. Organic acids not only have a significant effect on the sour taste of beer but also lower the pH of beer, which affects the quality and stability of beer flavour [50]. The concentration of citric acid in beers produced by non-Saccharomyces yeasts was 0.1 ± 0.0 g·L−1 (Table 6). Beer prepared with S. boulardii contained 0.3 ± 0.0 g·L−1 of citric acid and unlike beers fermented with four other non-Saccharomyces yeasts (K. lactis, K. marxianus and both P. manshurica strains), it also contained acetic acid (0.1 ± 0.0 g·L−1), which is not desirable. Unique mutations in S. boulardii cause an accumulation of higher amounts of acetic acid, which on the other hand might inhibit bacterial growth [51], but acetic acid drastically affects the taste of beer, imparting sharp acidity and vinegar notes when present in beer above the threshold concentration of 200 mg·L−1 [52,53].

3.7. Volatile Organic Compound HS-SPME-GC-MS Analysis

Among the most important factors influencing the organoleptic quality of beer is the presence of higher alcohols, esters, and carbonyl compounds. Our study revealed that during submerse fermentations, non-Saccharomyces yeasts of probiotic potential, Kluyveromyces lactis, K. marxianus, and both Pichia manshurica strains, displayed potential in the production of fermentation by-products, namely esters and higher alcohols, which is interesting from the brewer’s perspective.
The synthesis of higher alcohols via the Ehrlich pathway involves brewing yeasts absorbing amino acids from the wort, where the amino acids serve as carriers of essential amino groups that act as building blocks for forming yeast structural components, after which the remains of the amino acids (α-keto acids) are irreversibly converted to higher alcohols [54]. An increase in fermentation temperature strongly affects the transport of amino acids into the yeast cell, thus favouring an increase in higher alcohol production [55]. As the formation of higher alcohols is temperature-dependent, it also strongly influences final ester formation, where higher alcohols are necessary for ester formation [56]. Our study revealed that the yeasts P. manshurica, K. lactis, and K. marxianus were able to introduce higher alcohols such as 2-methyl-1-propanol, 2-methyl-1-butanol, and 3-methyl-1-butanol into the beer; however, the concentrations of these alcohols were several times lower in comparison with the fermentation led by the probiotic strain S. boulardii (Table 7). The authors of [14] worked with the probiotic strain of S. boulardii and described influence of the main fermentation parameters (temperature, pitch rate, wort composition) on the content of higher alcohols and esters, revealing that increasing fermentation temperature and pitch rate increases higher alcohol and ester formation.
Esters might be implemented into a beer during fermentation, including acetate esters (ethyl, 2-phenylethyl, and 3-methylbutyl acetate) and ethyl esters (ethyl hexanoate, octanoate, and decanoate) [57]. The formation of acetate esters involves higher alcohols, and the ethyl esters are formed by a condensation reaction of ethyl alcohol and acyl-CoA [58]. The final concentration of esters in beer is closely related to composition of used wort and fermentation conditions [59]. Ethyl acetate and 2-phenylethyl acetate were the only ethyl esters detected in beers prepared in this study (Table 7). According to [60], yeast K. marxianus and K. lactis might produce increased concentrations of volatile organic compounds such as esters and higher alcohols during the fermentation process.
The positive POF+ phenotype for all tested yeasts (Table 2) was supported by the presence of 4-vinylguaiacol (clove aroma) in the final beers (Table 7). Diacetyl (2,3-butanedione), an unwanted yeast metabolite (buttery aroma), was not detected in the final beers. These results favour the use of novel potential probiotic yeasts which might positively tailor the aromatic profile of final non-alcoholic beer (e.g., wheat-style beers) and boost its functionality as a novel beverage with health benefits.

4. Conclusions

In recent years, rising interest in producing functional beers using yeasts with potential probiotic attributes in the beverage industry has been taking place. In the presented study, we focused on the potentially probiotic yeasts Kluyveromyces lactis, K. marxianus, and Pichia manshurica and their application in non-alcoholic beer (NAB) production, using Saccharomyces boulardii as a control probiotic strain. The characterisation of yeast strains demonstrated survival in the simulated conditions of the human digestive tract (human body temperature and stomach acidic pH) after 24 h. On top of that, the tested yeasts were able to ferment the beer matrix (wort) and sustained different IBUs (iso-α-bitter acid concentrations) as a sole fermentation culture with targeted conditions to produce NABs. The stabilisation of beer achieved the inactivation of yeast, but the yeast cells remained intact—which might serve for the functionality of the beer as a postbiotic. The final NABs prepared using the non-Saccharomyces potential probiotic yeasts K. marxianus, K. lactis, and P. manshurica (first time used in the brewing) showed potential in tailoring a final beer sensory profile by producing higher alcohols (2-methyl-1-propanol, 2-methyl-1-butanol and 3-methyl-1-butanol, 2-phenylethanol,) and esters (ethyl acetate and ethyl hexanoate), as well as no acetic acid, making them a suitable alternative to the commercially available probiotic yeast S. boulardii. All tested yeast strains exhibited the production of 4-vinylguaiacol (clove) which was supported by a POF+ phenotype suited for wheat-style beer. This study provides insights into further applications in functional beer production using novel non-Saccharomyces potential probiotic yeast strains.

Author Contributions

Conceptualization, P.V., J.B. and P.D.; methodology, validation, P.V., J.B. and R.J.; formal analysis, P.V., J.B. and K.F.; investigation, J.B., P.V. and R.J.; resources, T.K., P.D. and D.Š.; data curation, P.V. and K.F.; writing—original draft preparation, P.V.; writing—review and editing, T.K. and K.F.; visualisation, K.F., J.B. and P.V.; supervision, P.D. and D.Š.; project administration, P.D. and D.Š.; funding acquisition, P.D. and D.Š. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences VEGA 1/0063/18, by Slovak Research and Development Agency APVV-22–0235 and Sport of the Slovak Republic within the Research and Development Operational Program for the project ‘University Science Park of STU Bratislava’, ITMS 26240220084.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Beverages 11 00140 g001
Figure 1. Temperature tolerance of tested yeasts in 2% YPD medium at three different temperatures (4, 20, and 37 °C) after 24 h by optical density (ΔOD600nm) measurements. Results are presented as average values of three OD600 nm measurements.
Figure 1. Temperature tolerance of tested yeasts in 2% YPD medium at three different temperatures (4, 20, and 37 °C) after 24 h by optical density (ΔOD600nm) measurements. Results are presented as average values of three OD600 nm measurements.
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Beverages 11 00140 g002
Figure 2. pH tolerance of tested yeasts in 2% YPD medium with different pH values (3–6) after 24 h at 37 °C by optical density (∆OD600nm) measurements. Results are presented as average value of three OD measurements.
Figure 2. pH tolerance of tested yeasts in 2% YPD medium with different pH values (3–6) after 24 h at 37 °C by optical density (∆OD600nm) measurements. Results are presented as average value of three OD measurements.
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Beverages 11 00140 g003
Figure 3. Iso-α-bitter acid tolerance of tested yeasts in 2% YPD medium with different IBU concentrations (10, 30, and 50) after 24 h at 25 °C by optical density (∆OD600nm) measurements. Results are presented as average value of three OD measurements.
Figure 3. Iso-α-bitter acid tolerance of tested yeasts in 2% YPD medium with different IBU concentrations (10, 30, and 50) after 24 h at 25 °C by optical density (∆OD600nm) measurements. Results are presented as average value of three OD measurements.
Beverages 11 00140 g003
Table 1. Yeast strains examined in this study with their abbreviations and brief characterisation.
Table 1. Yeast strains examined in this study with their abbreviations and brief characterisation.
YeastAbbreviationCharacterisation
Pichia manshurica 1 CCY * 039-063-001PM1Potential probiotic strain
Pichia manshurica 2 CCY * 039-063-004PM2Potential probiotic strain
Kluyveromyces lactis CCY * 026-012-002KLPotential probiotic strain
Kluyveromyces marxianus CCY * 029-008-010KMPotential probiotic strain
Saccharomyces cerevisiae var. boulardii HANSEN CBS ** 5926 (syn. S. boulardii)SBLControl probiotic strain
* CCY = Culture Collection of Yeasts (Bratislava, Slovakia); ** CBS = Central Bureau of Fungal Cultures (The Netherlands).
Table 2. Determination of saccharide fermentation tests, β-glucosidase activity, and phenolic off-flavour (POF) phenotype tests with studied yeasts after 24 h incubation at 25 °C.
Table 2. Determination of saccharide fermentation tests, β-glucosidase activity, and phenolic off-flavour (POF) phenotype tests with studied yeasts after 24 h incubation at 25 °C.
Yeast (Abbreviation)* Saccharide Fermentation** β-Glucosidase Activity*** POF Phenotype
GlucoseMaltoseLactose
Saccharomyces boulardii (SBL)++−positivePOF+
Pichia manshurica 1(PM1)+−−w/dPOF+
Pichia manshurica 2 (PM2)+−−w/dPOF+
Kluyveromyces lactis (KL)+−+positivePOF+
Kluyveromyces marxianus (KM)+−+positivePOF+
* “+”: positive formation of CO2—yeast was able to ferment saccharide, “−”: negative formation of CO2—yeast was unable to ferment saccharide; ** “w/d”: weak or delayed β-glucosidase activity; *** “POF+”: positive formation of phenolic off-flavours.
Table 3. Viability of tested yeasts after 24 h incubation of yeasts in a 2% YPD medium at three different temperatures (4 °C, 20, and 37 °C).
Table 3. Viability of tested yeasts after 24 h incubation of yeasts in a 2% YPD medium at three different temperatures (4 °C, 20, and 37 °C).
YeastS. boulardii (SBL)P. manshurica 1 (PM1)P. manshurica 2 (PM2)K. lactis (KL)K. marxianus (KM)
Viability at 4 °C83%27%30%35%37%
Viability at 20 °C97%82%83%80%85%
Viability at 37 °C98%97%98%96%96%
Table 4. Viability of tested yeasts after 24 h incubation at 37 °C in 2% YPD medium at pH 3 and 6.
Table 4. Viability of tested yeasts after 24 h incubation at 37 °C in 2% YPD medium at pH 3 and 6.
YeastS. boulardii (SBL)P. manshurica 1 (PM1)P. manshurica 2 (PM2)K. lactis (KL)K. marxianus (KM)
Viability at pH 369%17%16%21%18%
Viability at pH 697%84%86%83%87%
Table 5. Ethanol concentration and pH of the beer samples prepared from 8 °P Wort, fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalised at 400 MPa (3 min).
Table 5. Ethanol concentration and pH of the beer samples prepared from 8 °P Wort, fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalised at 400 MPa (3 min).
Sample
Basic Parameters8 °P WortSBLPM1PM2KLKM
Ethanol % (v/v)n.d.1.52 ± 0.060.04 ± 0.010.07 ± 0.010.13 ± 0.020.14 ± 0.01
pH6.00 ± 0.064.83 ± 0.025.80 ± 0.015.73 ± 0.025.41 ± 0.015.36 ± 0.03
“n.d.” = not detected. Values are presented as (average ± standard deviation) from three parallel analyses. Abbreviations of beer samples correspond to yeast abbreviation used for beer production. SBL = S. boulardii; PM1 and PM2 = P. manshurica 1 and 2; KL = K. lactis; KM = K. marxianus.
Table 6. Concentration of organic compounds (g·L−1) in beer samples prepared from 8 °P Wort fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalised at 400 MPa (3 min), as determined by HPLC-RID-DAD.
Table 6. Concentration of organic compounds (g·L−1) in beer samples prepared from 8 °P Wort fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalised at 400 MPa (3 min), as determined by HPLC-RID-DAD.
Sample
Compound (g·L−1)8 °P WortSBLPM1PM2KLKM
Glucose7.3 ± 0.2n.d.5.3 ± 0.25.4 ± 0.15.4 ± 0.15.4 ± 0.1
Maltose36.8 ± 0.524.1 ± 0.335.5 ± 0.335.1 ± 0.334.3 ± 0.335.5 ± 0.6
Glyceroln.d.1.2 ± 0.00.3 ± 0.10.5 ± 0.00.5 ± 0.00.5 ± 0.0
Citric acidn.d.0.3 ± 0.00.1 ± 0.00.1 ± 0.00.1 ± 0.00.1 ± 0.0
Acetic acidn.d.0.1 ± 0.0n.d.n.d.n.d.n.d.
Lactic acidn.d.n.d.n.d.n.d.n.d.n.d.
Abbreviations of beer samples correspond to yeast abbreviation used for beer production. SBL = S. boulardii; PM1 and PM2 = P. manshurica 1 and 2; KL = K. lactis; KM = K. marxianus. Values are presented as (average ± standard deviation) from three parallel analyses. “n.d.” = not detected.
Table 7. Concentration of VOCs (volatile organic compounds (μg·L−1)) of the beer samples prepared from 8 °P Wort fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalized at 400 MPa (3 min), as determined by HS-SPME-GC-MS.
Table 7. Concentration of VOCs (volatile organic compounds (μg·L−1)) of the beer samples prepared from 8 °P Wort fermented with tested yeasts (1 × 106 Cells·mL−1) at 20 °C (2 days) and maturated at 3 °C (3 weeks) and pascalized at 400 MPa (3 min), as determined by HS-SPME-GC-MS.
Beer Sample
Compound
(μg·L−1)		SBLPM1PM2KLKM
Ethyl acetate530.5 ± 85.818.6 ± 6.724.4 ± 7.9212.0 ± 6.3373.8 ± 54.1
2-Phenylethyl acetate73.1 ± 18.4n.d.n.d.358.1 ± 7.1166.7 ± 11.2
3-Methylbutyl acetaten.d.n.d.n.d.n.d.n.d.
2-Methyl-1-propanol981.4 ± 199160.6 ± 26.5983.5 ± 87.2371.1 ± 92.7212.4 ± 46.8
2-Methyl-1-butanol2867.3 ± 66.7363.5 ± 35.7828.2 ± 13.1803.5 ± 42.4488.2 ± 50.0
3-Methyl-1-butanol6125.8 ± 21.5615.0 ± 57.11195.5 ± 38.5992.8 ± 20.2775.2 ± 48.9
2-Phenylethanol7955.3 ± 163.11730.0 ± 220.42377.2 ± 78.91372.9 ± 93.21197.5 ± 112.1
Ethyl hexanoate274.2 ± 21.6162.9 ± 18.9146.0 ± 7.6139.0 ± 3.0149.4 ± 7.6
Ethyl octanoate307.5 ± 44.3n.d.n.d.n.d.n.d.
Ethyl decanoate366.1 ± 91.6n.d.n.d.n.d.n.d.
Hexanoic acid3855.8 ± 488.0332.4 ± 30.3304.3 ± 51.6239.1 ± 15.0274.7 ± 16.2
Octanoic acid2736.1 ± 484.2827.3 ± 140.6524.4 ± 50.7381.9 ± 23.8448.1 ± 15.2
Decanoic acid1420.8 ± 273.5n.d.n.d.n.d.n.d.
4-Vinylguaiacol3033.9 ± 48.3650.11 ± 46.4628.10 ± 36.6629.65 ± 44.7681.30 ± 50.1
Butane-2,3-dionen.d.n.d.n.d.n.d.n.d.
Abbreviations of beer samples correspond to a yeast abbreviation used for beer production. SBL = S. boulardii; PM1 and PM2 = P. manshurica 1 and 2; KL = K. lactis; KM = K. marxianus. Values are presented as (average ± standard deviation) from three parallel analyses. “n.d.” = not detected.
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Vaštík, P.; Brunner, J.; Jung, R.; Klempová, T.; Furdíková, K.; Šmogrovičová, D.; Dostálek, P. Functional Non-Alcoholic Beer Fermented with Potential Probiotic Yeasts. Beverages 2025, 11, 140. https://doi.org/10.3390/beverages11050140

AMA Style

Vaštík P, Brunner J, Jung R, Klempová T, Furdíková K, Šmogrovičová D, Dostálek P. Functional Non-Alcoholic Beer Fermented with Potential Probiotic Yeasts. Beverages. 2025; 11(5):140. https://doi.org/10.3390/beverages11050140

Chicago/Turabian Style

Vaštík, Peter, Ján Brunner, Rudolf Jung, Tatiana Klempová, Katarína Furdíková, Daniela Šmogrovičová, and Pavel Dostálek. 2025. "Functional Non-Alcoholic Beer Fermented with Potential Probiotic Yeasts" Beverages 11, no. 5: 140. https://doi.org/10.3390/beverages11050140

APA Style

Vaštík, P., Brunner, J., Jung, R., Klempová, T., Furdíková, K., Šmogrovičová, D., & Dostálek, P. (2025). Functional Non-Alcoholic Beer Fermented with Potential Probiotic Yeasts. Beverages, 11(5), 140. https://doi.org/10.3390/beverages11050140

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