Evaluation of Probiotic Saccharomyces boulardii Yeast as a Distillery Strain

Abstract

The probiotic properties of the yeast Saccharomyces boulardii are fairly well recognised, and research into the use of this strain in fermentation processes has been ongoing for several years. In this article, we have described the research results to evaluate the distillery potential of S. boulardii yeast. Compared to Ethanol Red and Thermosacc Dry yeast, the probiotic strain formed slightly different amounts of volatile compounds and fermented the available sugars less vigorously. The final ethanol concentration formed by the probiotic yeast was close to that observed for the distillery strains. Rye distillates with an alcohol content of 40% (v/v) obtained with S. boulardii yeast, according to the sensory panel, were distinguished by their delicately composed flavour and were rated better than distillates after fermentation by distillery yeast. The results are promising for the possibility of production of niche distillates using probiotic yeast.

1. Introduction

For centuries, humankind has used yeast to obtain products such as bread, kephir, wine, beer, and other traditionally fermented products. However, it is relatively recently that new remarkable properties of yeast as probiotic organisms have begun to be discovered. The only organism classified as yeast with proven probiotic activity is Saccharomyces cerevisiae var. boulardii. The discovery of this yeast species is linked to a trip by the French microbiologist Henri Boulard to Indochina (today, Vietnam, Laos, and Cambodia). At the time of his research, there was a cholera epidemic in that region. In 1920, Henri Boulard noticed that people who drank tea drink with the addition of lychee and mangosteen fruit peels did not contract cholera. He decided to isolate the yeast found on the surface of these fruits. The isolated organism was named S. boulardii and subsequently patented. Biocodex purchased the rights to the patent in 1947 [1]. Genetic studies to determine exact systematics have shown that S. boulardii shares more than 99% of its genome with S. cerevisiae, with the closest affinity being shown to be wine strains [2]. Due to this close genetic relationship, the name Saccharomyces cerevisiae varieties boulardii should be used for the correct terminology for this strain, or it should be noted in the text that the abbreviated name is used. The detailed genetic analysis revealed that there are groups of genes with increased copy numbers in the S. boulardii genome. These genes are responsible for encoding proteins that can be classified into two groups: stress resistance (HSP26, SSA3, SED1, HSP42, HSP78, PBS2) and protein synthesis (RPL31A, RPL41A, RPS24B, RPL2B, RSA3). The increased copy number of these genes may allow these yeasts to better adapt to stress conditions such as low pH [2]. The yeast S. boulardii is characterised by higher acetic acid production compared to the distiller strain Ethanol Red belonging to S. cerevisiae. This is due to the mutation and variable copy number of the SDH1 and WHI2 genes compared to S. cerevisiae. Increased acetic acid production at 37 °C correlates with antimicrobial activity [3].

The traditional classification of yeasts is based on biochemical criteria and morphological characteristics. In 2014, selected biochemical features of nine S. boulardii strains were compared—

Table 1. Assimilation of selected compounds by S. boulardii strains examined by Rajkowska and Kunicka-Styczyńska [4].

Compound	Strain
	Enterol® 250	Hamadin® N	Omniflora® Akut	Perenterol® Forte	Perocur® Forte	Santax® S	Yomogi®	MYA-796	MYA-797
D-glucose	+	+	+	+	+	+	+	+	+
Glycerol	+	+	-	+	+	+	-	+	+
D-galactose	+	-	-	-	-	-	-	-	-
D-cellobiose	+	-	-	-	+	-	-	-	-
D-maltose	+	+	+	+	+	+	+	+	+
D-sucrose	+	+	+	+	+	+	+	+	+
D-trehalose	-	-	-	-	+	-	-	+	-
D-raffinose	+	+	+	+	+	+	+	+	+

+—Positive, -—negative.

[4]. Two strains were from ATTC pure culture collection; the other seven were isolated from commercially available probiotic preparations. Faced with the compounds tested as potential carbon or nitrogen sources, none of the strains exhibited the ability to assimilate L-arabinose, L-xylose, xylitol, inositol, D-sorbitol, N-acetylglucosamine, D-lactose, D-melozitose, potassium nitrite, or ethylamine.

The term “probiotic” was first used in 1965 and means “for life.” In this context, a probiotic can be an organism or substance that benefits the host’s health. Since then, the need for a strict definition of the term has emerged in the scientific and legal world. In 2001, a definition emerged, created by WHO and FAO specialists, that states that a probiotic is a living microorganism that benefits the host’s health when administered in the correct dose [2]. There are several ways in which a probiotic can improve health, including modulation of the immune system, interaction with the brain–gut axis, antagonism towards pathogenic organisms, metabolic regulation, antitoxic properties, cell adhesion, mucin production, regulation of the microbiota, trophic actions, and modification of signalling pathways [5]. The probiotic effects of S. boulardii yeast can be divided into four main categories: trophic effect, immune system regulation, antimicrobial activity, and pathogen cell adhesion [6,7].

Initially, fermentation was mainly used to preserve food. The process also makes creating new products with specific taste qualities possible. However, more important, as well as more and more desirable in the modern world, are the health-promoting properties of food. As a result, pickled and fermented products are becoming increasingly popular. Increasing consumer awareness of the health-promoting properties of food has fostered research and the introduction of new organisms into industrial food production. In recent years, many publications have described using S. boulardii in beer production [8,9,10,11,12].

Díaz et al. [8] reported the usage of S. boulardii and S. cerevisiae yeasts in brewing as a control sample for beer production. The beers for which the probiotic yeast was used were characterised by more floral and fruity aromas than the control sample. These beers showed several positive features, such as lower turbidity (a characteristic that may allow the beer to be filtered more easily after fermentation), lower pH (this may have the effect of slowing down beer spoilage), and lower alcohol content (this may be beneficial for the production of low-alcohol beer). After 30 days of fermentation, second fermentation, and maturation, there were 6 × 10⁶ cells per mL in the beer, with a viability of 77% for all four hops used in the work [8].

Consumer trends point to a dynamic development of the low-alcohol drinks market. Global sales of low- and non-alcoholic beverages are growing year on year, and their share of the traditional alcohol market is steadily increasing. In particular, millennials and Generation Z are interested in low- or no-alcohol alternatives. Market research shows that more than 40% of 18–34-year-olds choose low- or no-alcohol drinks, looking for healthier alternatives to traditional alcohol [13,14]. The increasing interest in non-alcoholic or low-alcohol beers leads to attempts to use unusual microorganisms for their production. The literature reports the yeast S. boulardii as a potential beneficial component of the microflora of low-alcohol fermented beverages. The benefit of using this yeast is the possibility of creating a new health-promoting product. However, to do so, it would be necessary to determine whether the administration of S. boulardii yeast in beers has the same health benefits as classical, non-alcoholic, probiotic preparations. In addition, it would be necessary to determine how long such beers can be on the shelf and how storage time affects the viability of the yeast [12].

An interesting study on the preparation of low-alcohol sangria-type wines and ciders based on associated kefir cultures was published by Nikolaou et al. [15,16]. The literature also exemplifies the possible use of S. boulardii in yoghurts as a new way of administering probiotic yeast [17].

Distillers rely overwhelmingly on high-performance yeast strains supplied by global corporations. The use of these strains is due to the predictable fermentation, high yield, and ability to select the unique characteristics of the yeast to suit the technology and raw material used in the distillery. The purpose of the distillate created is also essential. For the production of pure vodkas, the rectified spirit is generally used, which should be characterised by a minimum of by-products. In contrast, for the technology of flavourful spirits such as whisky, rum, tequila, calvados, or plum brandy, producers base the fermentation of their mash, in part or whole, on the native microflora of the raw materials. One reason for this is economical, but it must be mentioned that the use of native microflora during the fermentation of fruit distillates results in the creation of unique taste and aroma characteristics of the distillates, which are desirable in the final products of this type [18,19,20,21,22,23,24,25].

The primary use of S. boulardii yeast to date has been to utilise its probiotic qualities for the composition of pharmaceutical preparations to improve the gut and general health of humans and animals. The few papers cited above address the use of probiotic yeast in producing beer, wine and other fermented products. We were unable to find reports on the evaluation of the distillery characteristics of this yeast strain, so we thought it would be interesting to determine the basic distillery features of this strain, such as the fermentation dynamics of grain mashes and molasses worts and the formation of volatile products affecting the sensory characteristics of distillates. From a scientific point of view, our results bring a comparison between the profiles of volatile compounds formed by the yeast Saccharomyces boulardii and traditional distillers’ S. cerevisiae strains sold under the commercial formulations Thermosacc Dry (TD) and Ethanol Red (ER), known for creating a balanced amount of fermentation by-products, but especially for the rapid and efficient fermentation of distillers’ mash made from starchy and fruity raw materials [25,26,27,28].

When we planned the research described in this article, we hoped to obtain results that would not only broaden the above-mentioned scientific knowledge but also hoped that similar work could demonstrate the new industrial potential of probiotic yeast in spirit distillate technology. We leave it to the reader to evaluate this assumption, together with our results and the final conclusions we describe.

2. Materials and Methods

2.1. Yeast Strains

The yeast used in this study was S. boulardii CNCM I-745 (SB) (Biocodex, Gentilly, France), and, as control samples, yeasts used in distilling were Thermosacc Dry (TD) (Lallemand Inc., Milwaukee, WI, USA) and Ethanol Red (ER) (Leaf by Lesaffre, France).

2.2. Activation of Yeast

We carried out the yeast activation according to the method we described earlier [29], using the parameters detailed below. The lyophilised S. boulardii was rehydrated in 150 mL of YPG broth (containing 10 g/L yeast extract, 20 g/L bactopeptone, and 20 g/L glucose, with a pH of 5.2 ± 0.1) that had been sterilised by autoclaving at 121 °C for 20 min. The yeast was then cultured at 32 ± 1 °C for 24 h using a reciprocal laboratory shaker set to 140 oscillations per min (Eberbach E5900, Belleville, NJ, USA). After cultivation, the yeast cell suspension was centrifuged (Laboratory Centrifuge MPW380R, Warsaw, Poland) at 5000× g for 10 min and washed twice with sterile 0.9% NaCl solution. The final yeast suspension’s dry matter (DM) content was measured using a spectrophotometer (Rayleight Analytical Instrument, Beijing, China) at 540 nm, based on a previously prepared standard curve. Two distillery yeast Thermosacc Dry and Ethanol Red preparations were used for reference. One hour before inoculation, the dried yeast cells of these strains were suspended in a sterile 0.9% NaCl solution, and the DM content was measured as described above for S. boulardii. The yeast strains were used in 0.9% NaCl suspensions at 1 g of DM per 1 L of juice for fermentation.

2.3. Substrate Preparation and Fermentation

Two molasses worts and two rye mashes were used for the evaluation of the fermentation properties of probiotic yeast: two molasses worts with total extracts of 10 °Blg (Balling degree) and 17.5 °Blg and two rye mashes with total extracts of 10 °Blg and 17.5 °Blg. Additionally, the 21.5 °Blg rye mash was fermented to prepare the distillates.

2.3.1. Preparation and Fermentation of Molasses Worts

For the preparation and fermentation of the molasses wort, we used a modification of the procedure described by Dziugan et al. [30], with details as described below. Sugar beet molasses with an extract content (obtained from a sugar factory located in Dobrzelin, Poland) of 75.6 °Blg was diluted to yield 4.5 L containing 10 °Blg or 17.5 °Blg. To the diluted molasses, 0.2 g/L of ammonium diphosphate were added, then using 10% sulphuric acid (IV), the pH was set to 5.0 ± 0.1. The medium prepared this way was bottled at 500 mL into flasks of 1 L capacity. The medium was inoculated with previously prepared yeast suspensions. The yeast inoculum of 1 g DM /1 L of medium was used. After thorough mixing, the outlet of the flask was closed with a stopper and a tube containing glycerol that prevented sample contact with oxygen from the air. The inoculated worts were placed in a thermostatic room with a constant temperature of 32 ± 1 °C. Fermentations were carried out for 4 d for substrates with a sugar content of 10 °Blg, while for substrates containing 17.5 °Blg, fermentations were carried out for 5 d.

2.3.2. Preparation and Fermentation of Rye Mashes

For the preparation and fermentation of the grain mash, we used a modification of the procedure described by Balcerek et al. [31], with details modified as described below. Rye flour type 720 (Szczepanki, Poland) was used as a raw starch material. For the starch hydrolysis, the pressureless liberation of starch (PLS) technology was used to prepare rye mashes by heating the mixture to 90 °C and adding amylolytic enzymes. LpHera (exhibiting α-amylase activity; Novozymes, Aesh, Switzerland) and Saczyme Plus (exhibiting glucoamylase activity; Novozymes, Aesh, Switzerland) enzyme preparations were used. The 4 kg of flour were suspended in tap water at a ratio of 3.5 L of water per 1 kg of flour. Once the flour was entirely homogenously mixed in 14 L of water, the whole mixture was heated with continuous stirring to a temperature of 90 °C. For starch liquefaction (partial hydrolysis of amylose and amylopectin chains), the enzyme preparation LpHera was added (0.5 mL of preparation per 1 kg of starch). The temperature was maintained for 1 h, then lowered to 60 °C. Then, the glucoamylase preparation Saczyme Plus (0.72 mL per kg of starch) was added to induce complete starch degradation into glucose subunits. The whole medium was cooled to 30 °C. The medium thus prepared was diluted to yield a final of 10 °Blg or 17.5 °Blg. Subsequently, 0.2 g/L of ammonium diphosphate were added, and then, using sulphuric acid (IV), the pH was set to 5.0 ± 0.1. Substrate partitioning and inoculation proceeded in the same way as for molasses media. The fermentation temperature was the same as for molasses worts, while its time was 4 d for 10 °Blg wort and 5 d for 17.5 °Blg.

The rye mash intended to prepare the distillates was prepared using the same PLS method described above. The resulting mash was made up to a volume of 15 L (corresponding to a density of 21.5 °Blg) and then divided into three portions of 5 L each, placed in fermentation containers. A total of 0.2 g/L of phosphate diammonium were added, pH 5.0 ± 0.1. Fermentation was carried out for 7 d at 32 ± 1 °C.

To determine the fermentation dynamics, each fermenting sample was weighed after inoculation with the inoculum, followed by weight measurements during fermentation. It was assumed that the mass loss mainly resulted from the carbon dioxide release formed by yeast during the sugar-to-ethanol bioconversion process. Fermentation dynamics were presented as an average weight loss during the mash’s fermentation.

The samples were fermented in 3 replicates. After fermentation, small samples (ca. 100 mL) were taken and stored in a −20 °C freezer to preserve them for later analyses.

2.4. HPLC Analysis

HPLC analysis of fermented media was performed as described in Balcerek et al. [32] using an Agilent 1260 Infinity apparatus (Agilent Technologies, Santa Clara, CA, USA) equipped with a Hi-Plex H column (7.7 × 300 mm, 8 μm; Agilent Technologies, USA) and an RI detector at 55 °C. The column temperature was 60 °C, and 5 mM H₂SO₄ was used as a mobile phase at a flow rate of 0.7 mL/min with a sample injection of 20 μL.

2.5. Distillation of Rye Mashes

Each fermented rye mash (starting extract 21.5 °Blg) was distilled using a 6 L glass round-bottomed flask, a heating mantle, and a Liebig condenser. Distillation was carried out until an alcohol concentration of 1% was reached in the current distillate. On completion of the distillation, three distillates were obtained, each in a volume of around 1.4 L, which were characterised by an alcohol content [v/v] of 34.73% for S. boulardii, 4.55% for TD, and32.90% for ER. Alcohol concentrations were routinely measured at 20 °C using an areometer calibrated in % alcohol by volume.

The fortification of the distillates was carried out by re-distillation using an apparatus that consisted of a heating mantle, a 2 L round-bottomed flask, a bi-rectifier (dephlegmator according to Golodetz), and a Liebig condenser [33]. The first 50 mL of the distillate were intentionally removed to separate some low-boiling compounds (head fraction). The final distillates obtained were characterised by an alcohol content [v/v] of 57.78% for S. boulardii, 56.99% for TD, and 55.44% for ER. Areometric alcohol measurements were also randomly confirmed using an Anton Paar digital oscillating densitometer DMA 1001 (Graz, Austria) calibrated in % alcohol by volume [34].

2.6. Gas Chromatography Analysis of the Fermented Media and Distillates

GC analysis of the fermented media was performed using a GC apparatus Agilent 7890A (Agilent Technologies, Santa Clara, CA, USA) with a mass spectrometer Agilent MSD 5975C (Agilent Technologies, Santa Clara, CA, USA). An HP-5 MS capillary column (30 m, 0.25 µm film, and 0.25 mm i.d.) was used to separate compounds. The procedure was described in detail by Balcerek et al. [31].

2.7. Sensory Analysis

Samples of three rye distillates obtained after fermentation of rye mash with an initial extract of 21.5 °Blg by SB, TS, and ER strains were subjected to sensory analysis. The distillates were diluted to a strength of 40% (v/v). The study was conducted by a panel of six experts knowledgeable about production and the requirements for alcoholic beverages. Tasters were asked to give their colour, clarity, aroma, and taste ratings. Using Buxbaum’s positive ranking sensory evaluation method, respondents could assign the following points in each category: colour from 0 to 2, clarity from 0 to 2, aroma from 0 to 4, and taste from 0 to 12. The maximum number of points in the final evaluation was 20 [35]. The samples were coded to maintain complete objectivity. The scoring was complemented by allocating points in the “overall, total taste, and aroma experience” category. The available rating scale for this was from 0 to 10. The question aimed to determine which distillates tested would be the respondents’ choice as the alcoholic beverage they would most like to return to.

2.8. Statistical Analysis

The data presented here are average results. The Shapiro–Wilk and Bartlett’s tests were used to analyse fermentation dynamics and HPLC results. The Kruskal–Wallis rank-sum test and Dunn’s post hoc test with Holm’s correction were used to determine the presence of statistically significant differences. Calculations were performed using the R programme.

To determine the effect of the yeasts (SB, TD, and ER) and the initial extract (10% and 17% w/w) on the concentration of volatile compounds in the fermented molasses wort, two-factor analysis of variance (ANOVA) and Tukey’s multiple comparisons test were performed to assess the significance of the effect of individual variables and their interactions. Data obtained from the quantitative analysis of volatile compounds, including esters, higher alcohols, and carbonyl compounds, were used for the study.

3. Results and Discussion

3.1. Fermentation Dynamics

3.1.1. Fermentation Dynamics of Molasses Wort

Fermentation dynamics are expressed as weight loss during fermentation. The results obtained for the fermentation of 10 °Blg molasses wort show significant differences in the onset of the process occurring between the organisms tested (Figure 1). The fastest entry into the logarithmic process phase characterised the TD strain. At the same time, this yeast achieved the highest final weight loss (14.4 g). As the end of fermentation approached, a reduction in differences was observed for all yeasts tested. The final measurements showed similar results for all strains. The difference in end-weight loss between TD and ER strains was 1 g, while the difference between ER and SB strains was 0.4 g. These results show a prolonged adaptation phase of the SB yeast compared to the control strains.

To highlight possible differences occurring due to the increased wort density, the fermentation dynamics of the 17.5 °Blg molasses wort were evaluated (Figure 2). During the initial fermentation period, the TD yeast showed the fastest exit into the logarithmic phase of fermentation dynamics. The other yeasts, SB and ER, showed an extended adaptation phase compared to the TD strain. As fermentation progressed, the results for all yeast tested approached each other to produce similar results on the final day. The highest final weight loss was recorded for the TD strain (26.1 g). A total weight loss of 2.17 g less was recorded for the ER yeast, while the samples fermented by the SB yeast lost 3.2 g less than those using the ER yeast.

These results show that as the density of the wort increased, the differences between the tested strains became more pronounced. The highest results were obtained for both molasses worts for both TD yeasts (Figure 1 and Figure 2). It is also apparent that, of the strains tested, the SB yeast showed the worst adaptation to the fermentation of molasses mash.

3.1.2. Fermentation Dynamics of Rye Mash

The fermentation dynamics data obtained for the 10 °Blg rye mash (Figure 3) indicate that as in the cases of molasses worts (Figure 1 and Figure 2), TD yeast had the shortest adaptation phase compared to the other strains tested. However, the highest final weight loss in the 10 °Blg rye mash was noted for the SB yeast and was 19.5 g. The difference between the total weight loss of samples fermented by SB yeast and by TD yeast was 0.6 g, while the difference in final weight loss between TD- and ER-fermented worts was 0.1 g.

For the TD strain, the final weight loss of the molasses wort was 14.4 g, while for SB yeast, the loss of the rye mash mass was 19.5 g. The difference between worts with the same sugar content shows that all strains tested had a higher fermentation dynamic on the rye mash.

The results of the fermentation dynamics obtained during the fermentation of the 17.5 °Blg rye mash presented above follow a similar pattern to the results obtained for 10 °Blg rye mash (Figure 3). The highest total mass loss result was again observed for SB yeast (34.2 g). The difference between the highest result and the second highest result obtained for the ER strain was 0.6 g, while the difference between the ER and TD strains was 0.5 g.

A comparison of the results obtained in worts of the same density (Figure 1, Figure 2, Figure 3 and Figure 4) again shows that all of the tested yeasts exhibited higher fermentation dynamics on the rye mash than on the molasses substrate.

The results show that SB yeast exhibited better rye mash fermentation than when molasses worts were used. Regardless of the substrate, all the results show that SB yeast exhibited a prolonged adaptation phase compared to the other strains. Prolonging the adaptation phase may lead to an increased risk of infection and increase the time required to fully ferment the sugars available in the mash. A potential solution to this problem could be to increase the inoculum to inoculate the medium to be fermented. Another possible solution could be to inoculate the yeast earlier, in a smaller volume of wort, which will then be destined for fermentation in a larger volume. This may reduce the shock to the yeast, thus leading to a shorter adaptation phase.

When comparing the fermentation dynamics of molasses wort and rye mash, it should be noted that the rye mash probably contained considerably more yeast nutrients, such as amino acids or vitamins, compared to the relatively simple and “synthetic” composition of molasses wort.

Analyses of the results suggest that S. boulardii yeasts must be tested on specific substrates, which would later be used to produce alcoholic beverages on an industrial scale.

3.2. HPLC Analysis of Fermented Media

3.2.1. Results of HPLC Analysis of Molasses Worts After Fermentation

One of our research aims was to assess probiotic yeasts’ ability to ferment viable distillery substrates. We chose molasses wort and rye wort as examples, and distillers’ TD and ER strains were used as reference strains. In

Table 2. Results of the HPLC analysis of 10 °Blg molasses wort after fermentation [g/L].

Compound	Yeast
	SB	ER	TD
Citric acid	0.33 ± 0.04 a	0.23 ± 0.01 b	0.23 ± 0.01 b
Glucose	0.77 ± 0.14 a	0.06 ± 0.01 b	0.04 ± 0.01 b
Malic acid	0.65 ± 0.07 b	0.81 ± 0.09 a	0.78 ± 0.11 ab
Fructose	4.89 ± 0.76 a	2.47 ± 0.13 b	0.15 ± 0.11 c
Succinic acid	0.3 ± 0.1 a	0.2 ± 0.02 b	0.19 ± 0.02 b
Lactic acid	1.76 ± 0.18 a	1.63 ± 0.07 a	1.77 ± 0.18 a
Glycerol	2.83 ± 0.27 a	2.99 ± 0.13 a	2.92 ± 0.07 a
Acetic acid	1.07 ± 0.16 a	1.03 ± 0.05 a	1.0 ± 0.05 a
Ethanol	29.93 ± 0.65 a	31.38 ± 1.91 a	31.81 ± 1.33 a

^a–c—Mean values in the rows marked by different letters are significantly different.

,

Table 3. Results of the HPLC analysis of 17.5 °Blg molasses wort after fermentation [g/L].

Compound	Yeast
	SB	ER	TD
Citric acid	0.31 ± 0.06 a	0.36 ± 0.01 a	0.33 ± 0.01 a
Glucose	7.16 ± 1.13 a	0.95 ± 0.14 a	0.24 ± 0.03 b
Malic acid	0.59 ± 0.14 b	1.06 ± 0.09 b	1.54 ± 0.12 a
Fructose	22.28 ± 1.36 a	10.97 ± 1.29 a	1.43 ± 0.26 b
Succinic acid	0.29 ± 0.01 b	0.33 ± 0.02 ab	0.4 ± 0.03 a
Lactic acid	3.13 ± 0.05 a	3.06 ± 0.05 a	3.11 ± 0.04 a
Glycerol	4.98 ± 0.23 b	5.77 ± 0.01 a	5.64 ± 0.15 a
Acetic acid	2.12 ± 0.06 a	2.05 ± 0.04 a	1.83 ± 0.31 b
Ethanol	46.04 ± 2.33 c	53.41 ± 1.52 b	59.67 ± 0.82 a

^a–c—Mean values in the rows marked by different letters are significantly different.

,

Table 4. Results of the HPLC analysis of 10 °Blg rye mash after fermentation [g/L].

Compound	Yeast
	SB	ER	TD
Maltotriose	0.47 ± 0.02 a	0.55 ± 0.09 a	0.48 ± 0.05 a
Maltose	0.74 ± 0.08 a	0.26 ± 0.03 b	0.56 ± 0.1 ab
Citric acid	0.97 ± 0.03 a	0.89 ± 0.08 a	0.9 ± 0.16 a
Glucose	0.2 ± 0.02 a	0.12 ± 0.03 b	0.14 ± 0.02 b
Xylose	0.31 ± 0.05 b	0.58 ± 0.04 a	0.44 ± 0.07 a
Arabinose	0.11 ± 0.02 a	0.12 ± 0.008 a	0.05 ± 0.01 b
Succinic acid	0.74 ± 0.04 b	0.93 ± 0.03 a	0.9 ± 0.07 a
Glycerol	4.92 ± 0.07 a	5.59 ± 0.15 a	5.42 ± 0.29 a
Acetic acid	0.05 ± 0.003 a	0.04 ± 0.001 ab	0.03 ± 0.008 b
Propylene glycol	0.01 ± 0.002 a	0.01 ± 0.002 a	0.01 ± 0.001 b
Ethanol	37.39 ± 0.12 b	38.51 ± 1.15 a	37.38 ± 0.08 b

^a,b—Mean values in the rows marked by different letters are significantly different.

and

Table 5. Results of the HPLC analysis of 17.5 °Blg rye mash after fermentation [g/L].

Compound	Yeast
	SB	ER	TD
Maltotriose	0.76 ± 0.05 b	0.76 ± 0.02 b	0.85 ± 0.06 a
Maltose	0.58 ± 0.03 b	0.49 ± 0.04 b	0.39 ± 0.04 a
Citric acid	1.56 ± 0.04 a	1.68 ± 0.29 a	1.54 ± 0.15 a
Glucose	0.3± 0.03 a	0.22 ± 0.041 b	0.21± 0.01 b
Xylose	0.36 ± 0.03 c	0.6 ± 0.07 b	1.14 ± 0.21 a
Arabinose	0.22 ± 0.04 a	0.2 ± 0.03 a	0.21 ± 0.02 a
Succinic acid	1.025 ± 0.09 c	1.26 ± 0.09 b	1.47 ± 0.09 a
Glycerol	8.23 ± 0.13 b	8.89 ± 0.33 a	8.37 ± 0.23 b
Acetic acid	0.04 ± 0.001 b	0.13 ± 0.006 a	0.032 ± 0.002 b
Propylene glycol	0.02 ± 0.002 b	0.03 ± 0.003 a	0.02 ± 0.003 b
Ethanol	66.37 ± 0.77 a	63.12 ± 0.98 b	65.08 ± 1.26 ab

^a–c—Mean values in the rows marked by different letters are significantly different.

, we present the HPLC results of the mash after fermentation.

The only sugars tested in molasses worts were glucose and fructose. The highest glucose concentration of 0.77 g/L was recorded for the SB-fermented samples. A comparison of the results for the amount of glucose in the wort shows statistically significant differences between the samples using SB yeast and all control strains (ER, TD strains). Also, for fructose, SB fermentation resulted in the highest concentration of this sugar, at 4.89 g/L (Table 2). Statistical analysis of the fructose concentration shows a significant difference between the results obtained for SB and ER strains, with a disproportion of 2.42 g/L to the disadvantage of SB yeast. The poorest sugar assimilation by S. boulardii yeast sugars should be translated into reduced ethanol production. Despite this, no statistically significant differences in ethanol content were detected in the samples. The highest ethanol concentration was recorded in the sample fermented with TD yeast (31.81 g/L). Analysis of organic acids reveals that on the wort in question, SB yeast produced the most acid: citric acid (0.33 g/L), succinic acid (0.3 g/L), and acetic acid (1.07 g/L). Statistical differences between S. boulardii and the reference strains occurred for citric acid and succinic acid, but none were detected between strains for lactic acid content. Also, for glycerol, no statistical differences were detected between yeasts. The lowest amount of glycerol was detected for SB yeast (2.83 g/L).

Increasing the wort’s density makes highlighting potential differences in the production of individual compounds between strains possible. These discrepancies can be seen, among others, in the ethanol content, where the highest content of this compound on a molasses wort of 17.5 °Blg was measured for TD yeast—59.67 g/L (Table 3). Statistical analysis for ethanol revealed statistically significant differences between all tested yeasts. Of these, the smallest amount of ethanol was recorded for strain SB; the difference in ethanol concentration between this strain and TD was 13.63 g/L.

Statistically significant differences can also be seen in the residual sugar content after fermentation. The highest concentrations of both glucose and fructose were measured for SB yeast. These amounted to 7.16 g/L for glucose and 22.28 g/L for fructose. The lowest amounts of both sugars were observed for TD yeast samples. The remaining glucose for this strain was 0.24 g/L, and for fructose was 1.43 g/L.

For S. boulardii-fermented samples, the highest concentrations of lactic acid (3.13 g/L) and acetic acid (2.12 g/L) were noted. The citric acid content for all samples was 0.31–0.36 g/L. Similarly, in the case of succinic acid, the concentration for SB and ER yeast samples was close to 0.31 g/L. In the case of malic acid, the highest amount was recorded for the TD-fermented sample at 1.54 g/L.

Analysis of the glycerol content of the samples tested shows statistically significant differences between the results obtained for SB yeast and the reference strains (ER, TD). The lowest amount of this compound was recorded for probiotic yeast at a concentration of 4.98 g/L, while the other two strains obtained results close to 5.7 g/L.

A comparison of the results obtained on molasses worts allowed for an assessment of the increase in ethanol production depending on the extract (Table 2 and Table 3). Of the yeast tested, the smallest increase in ethanol production was recorded for the SB strain at 34.99%. The increase in this crucial compound for the other two yeasts was ER 42.25% and TD 47.70%. In both worts, the highest concentration of sugars remaining after fermentation was noted for the probiotic strain. Organic acid analysis shows that SB yeast produced the highest amounts of lactic and acetic acids in both worts (10 and 17 °Blg). This strain also produced the lowest glycerol in both worts, but in the molasses wort only of 17.5 °Blg; the difference in the content of this compound between the probiotic and the reference strain was statistically significant.

The results obtained in the molasses worts show that SB yeast manifested an underproduction of ethanol compared to the control distillers’ yeasts ER and TD. The lack of fructose assimilation capacity of the SB yeast strain tested can explain these results. The results align with the literature on individual SB yeast strains’ ability to assimilate compounds [4]. Literature data also report that S. boulardii yeast isolated from soya paste could assimilate sucrose and even starch [36]. The lack of fructose fermentation by the strain used in our study, combined with the literature data cited, highlights the need to carefully combine the strain with a specific medium to maximise the effects of biomass growth, or, as in our case, fermentation and ethanol formation.

Although it is difficult to find in the literature results of studies planned under conditions closely correlated to ours, we consider that the results of ethanol produced by us are comparable to those presented in the literature. In the case of the 17.5 °Blg molasses wort, the maximum ethanol concentration obtained by us was 59.67 g/L, which corresponds quite closely to the data presented by Beigbeder et al. [28], who, in the case of beet molasses wort fermentation, using the Thermosacc strain, obtained about 55 g/L ethanol after 120 h of fermentation of a wort containing 125 g/L sugars. For a wort containing 225 g/L sugars, they obtained about 90 g/L ethanol, with further increases in the concentration of sugars of up to 325 g/L, resulting in a decrease in the amount of ethanol obtained (75 g/L). Barbosa et al. [37] reported obtaining about 90% attenuation of sugarcane molasses wort (22 and 30% extract) using PE-2 and CAT 1 strains belonging to Saccharomyces cerevisiae. Interesting results after fermentation of 25 °Brix cane molasses wort were described by Hawaz et al. [38] for the strain Meyerozyma caribbica. After 72 h of fermentation, they recorded 56 g/L of ethanol produced. Raharja et al. reported 6.55 to 9.56% (v/v) ethanol after 54 h of fermentation of sugarcane wort for worts with initial extract contents of 20, 25, and 30% (w/v), respectively [39].

3.2.2. Results of HPLC Analysis of Rye Mash After Fermentation

All strains utilised maltotriose from 10 °Blg rye mash to a similar extent. In the case of maltose, the highest residual amount was recorded for SB yeast (0.74 g/L), while the highest utilisation of this sugar was observed for ER yeast, with 0.26 g/L remaining. Similarly, the highest amount of residual sugar was recorded for the probiotic strain for glucose. The results for the other two yeasts were significantly lower. The opposite results were observed for xylose, where for mash fermented with S. boulardii the lowest concentration (0.31 g/L) of this sugar was assayed. Significantly higher xylose concentrations were observed for both ER and TD reference strains. The results for arabinose concentrations in mashes fermented by strains SB and ER were not statistically different but were higher than those obtained for TD yeast (0.05 g/L).

The citric acid concentration varied between 0.89 and 0.97 g/L. The control strain used resulted in statistically similar succinic acid content, close to 0.91 g/L. In comparison, the same acid in the medium fermented by probiotic yeast was only 0.74 g/L, significantly different from the other two.

The highest amount of ethanol was noted for the ER yeast, at 38.51 g/L, while for SB and TD, this value was similar and close to 37.4 g/L. Compared to the results obtained after fermentations of 10 °Blg molasses, higher ethanol concentrations were observed for all yeasts. The difference in ethanol production when comparing the 10 °Blg molasses and the 10 °Blg rye wort medium was for strain SB, with 7.45 g/L; strain ER, with 7.13 g/L; and strain TD, with 5.57 g/L (Table 2 and Table 4).

Maltose and maltotriose are sugars formed during the hydrolysis of starch of rye mash. The highest amount of maltotriose remaining in the 17.5 °Blg rye mash (Table 5) was recorded after fermentation with TD yeast and was 0.85 g/L. At the same time, in the other two cases (ER and SB), this result was close to 0.75 g/L, and no statistically significant difference was observed between these two strains.

The highest amount of maltose remaining in the mash was recorded for SB yeast, at 0.58 g/L, while the lowest amount was observed for the sample using TD yeast, at 0.39 g/L. In the case of xylose in the 17.5 °Blg rye mash, as for the 10 °Blg mash from the same raw material, the lowest amount of this compound was observed after fermentation by the SB strain. The results of residual arabinose concentrations oscillated around 0.21 g/L and were not statistically different for the strains tested.

Comparing the amount of ethanol produced, it is apparent that the highest amount was produced by SB yeast (66.37 g/L). The least amount of ethanol (63.12 g/L) was produced during the fermentation of this mash by ER yeast, which at the same time showed the highest amount of glycerol produced (8.89 g/L). The difference in the ethanol produced between the 17.5 °Blg rye mash and the 17.5 °Blg molasses wort was, in favour of the rye mash, for the probiotic yeast, at 20.32 g/L, while for reference strains, it was 9.71 g/L for ER and 5.4 g/L for TD. As in the case of comparing the fermentation effects of a 10 °Blg rye mash to a 10 °Blg molasses wort, the most significant increase in ethanol production in the rye mash was observed for the SB yeast.

Similarly to the 17.5 °Blg molasses medium, SB yeast recorded the lowest glycerol content in both rye mashes: 4.92 g/L for the 10 °Blg rye mash and 8.23 g/L for the 17.5 °Blg rye mash.

3.2.3. Results of HPLC Analysis of 21.5 °Blg Rye Mash After Fermentation

After successful fermentation of the 17.5 °Blg rye mash, we decided to ferment in a denser mash (21.5 °Blg). The main objective was to prepare larger mash volumes so that we could successfully obtain sufficient distillates for organoleptic evaluation, but at the same time, we also subjected these mash samples to chromatographic analysis to scientifically complement the results from the 10 °Blg and 17.5 °Blg wort series. We also hoped that at this concentration we might be able to observe the adverse effects of increased osmotic pressure on the strains tested, because it should be noted that usually the starting density of grain worts in Polish distilleries does not exceed 18 °Blg, mainly due to the extended time needed for complete fermentation (as usually the density of worts is set so that complete fermentation occurs within 72 h).

SB yeast recorded the highest ethanol content after 21.5 °Blg rye mash fermentation (89.4 g/L), but very close concentrations (below the statistical difference) were also noted for reference strains. When analysing the sugars remaining in the medium after fermentation, no statistically significant differences were observed for all the compounds tested.

No statistically significant differences were observed for glycerol either. The highest concentration was observed for the ER yeast, at 11.73 g/L, while the lowest amount was recorded for the SB strain, with a difference of 1.79 g/L. These results align with those previously discussed for rye media (Table 4 and Table 5). The lower glycerol concentration in the SB strain samples may also be related to this yeast’s ability to assimilate glycerol [4]. This ability may also answer the question of why, despite similar amounts of sugars at the end of fermentation in the 17.5 °Blg and 21.5° Blg rye mashes, the SB yeast was characterised by a higher ethanol content than the ER and TD strains (Table 5 and

Table 6. HPLC results for 21.5 °Blg rye mash after fermentation [g/L].

Compound	Yeast
	SB	ER	TD
Maltotriose	0.75 ± 0.01 a	0.78 ± 0.04 a	0.88 ± 0.05 a
Maltose	0.64 ± 0.005 a	0.58 ± 0.05 a	0.57 ± 0.11 a
Citric acid	0.84 ± 0.001 a	0.71 ± 0.2 a	0.93 ± 0.01 a
Glucose	0.17 ± 0.007 a	0.11 ± 0.007 a	0.19 ± 0.04 a
Xylose	1.32 ± 0.02 a	1.04 ± 0.02 a	1.38 ± 0.03 a
Arabinose	0.13 ± 0.01 a	0.15 ± 0.002 a	0.17 ± 0.05 a
Succinic acid	1.68 ± 0.002 a	1.87 ± 0.03 a	1.92 ± 0.1 a
Glycerol	9.94 ± 0.006 a	11.73 ± 0.23 a	10.8 ± 0.04 a
Acetic acid	0.13 ± 0.0001 b	0.34 ± 0.0001 a	0.13 ± 0.001 b
Propylene glycol	0.04 ± 0.003 a	0.06 ± 0.001 a	0.04 ± 0.005 a
Ethanol	89.4 ± 0.09 a	88.15 ± 0.14 a	88.57 ± 0.08 a

^a,b—Mean values in the rows marked by different letters are significantly different.

).

In the case of rye mash, all assayed compounds assimilated by the two control strains were probably also assimilated by the SB yeast. This may also be supported by the results of the ethanol content, which were similar for all strains tested.

The combination of rye flour medium and yeast strains we used made it very difficult to find directly correlating results in the literature; however, we found several entries describing the ethanol formation efficiency of rye mash by Ethanol Red yeast. In our experiments, the maximum ethanol concentration after fermentation of the 21 °Blg mash was 98.4 g/L, which corresponds to the results presented by Pielech-Przybylska et al. [40], who, for a rye mash of 28.5% (w/v), after 72 h fermentation by the Ethanol Red strain recorded a final ethanol concentration range of 9.18 to 11.71% (v/v). Balcerek et al. [32], for a mash of a mixture of rye and rye grain malt containing 164.4–182.5 g extract in 1 L, after 72 h fermentation by the Ethanol Red strain determined up to 69.4 g/L ethanol. Similar results after taking into account the theoretical fermentation yield of starch to ethanol were reported by Strąk et al., who, for a rye mash with an initial extract content in the range of 21–25%, observed an ethanol yield of up to 37.4 L from 100 kg of rye with 68.5% starch [41].

3.2.4. Results of GC Analysis of Molasses Worts and Rye Mashes After Fermentation

Table 7. Summary of the GC analysis of fermented sugar beet molasses worts.

Compound Name	SB		TD		ER		p-Value Significance Codes 1
	10% (w/w)	17% (w/w)	10% (w/w)	17% (w/w)	10% (w/w)	17% (w/w)
	Mean	Mean	Mean	Mean	Mean	Mean	Yeast Strain	Extract	Yeast Strain × Extract
Isobutyraldehyde, mg/L	0.56 d	0.96 c	2.49 a	2.34 a	1.04 c	1.57 b	***	°	***
Diacetyl, mg/L	0.48 cd	1.65 a	0.69 b	0.28 e	0.34 de	0.57 bc	*	°	***
2-Methyl-1-propanol, mg/L	27.99 e	54.34 c	83.33 b	97.42 a	47.03 d	44.11 d	***	°	***
3-Methyl-1-butanol, mg/L	5.69 c	116.96 b	127.40 a	126.55 a	56.25 c	61.99 c	***	°	***
2-Methyl-1-butanol, mg/L	42.06 e	48.60 d	165.08 a	124.25 b	60.34 c	59.21 c	***	°	***
3-Methylbutyl acetate, mg/L	0.40 b	0.25 c	0.45 a	0.43 a	0.14 d	0.13 d	***	°	***
2-Methylbutyl acetate, mg/L	0.15 c	0.05 d	0.30 a	0.21 b	0.06 d	0.05 d	***	°	***
2-Phenylethanol, mg/L	18.76 d	24.23 c	29.79 b	46.64 a	28.46 bc	24.42 c	***	°	***
Phenethyl acetate, μg/L	57.28 a	38.39 b	56.20 a	40.16 b	17.36 c	16.54 c	***	°	***
Carbonyl compounds, mg/L	5.56 e	7.46 d	25.14 a	20.64 b	7.44 d	8.76 c	***	°	***
Higher alcohols, mg/L	145.78 e	244.12 c	405.64 a	394.87 b	192.08 d	189.74 d	***	°	***
Esters, mg/L	19.11 c	28.46 b	31.21 b	48.67 a	14.72 c	16.56 c	***	°	***

^a–e—Mean values in the rows marked by different letters are significantly different (two-way ANOVA, p < 0.05); ¹ significance codes: 0 < *** < 0.001; and 0.01 < * < 0.05 < ° < 1.

and

Table 8. Summary of the GC analysis of fermented rye mashes.

Compound Name	SB		TD		ER		p-Value Significance Codes 1
	10% (w/w)	17% (w/w)	10% (w/w)	17% (w/w)	10% (w/w)	17% (w/w)
	Mean	Mean	Mean	Mean	Mean	Mean	Yeast Strain	Extract	Yeast Strain × Extract
Ethyl formate, mg/L	0.08 d	0.18 a	0.09 d	0.10 c	0.08 d	0.14 b	°	***	***
Ethyl acetate, mg/L	3.78 c	8.63 a	3.51 c	7.78 b	3.82 c	8.49 a	°	***	***
2-Methyl-1-propanol, mg/L	69.99 d	92.79 b	89.65 b	103.97 a	79.27 c	88.42 b	*	***	***
1,1-Diethoxyethane, μg/L	9.17 d	57.99 b	5.33 d	26.97 c	6.17 d	142.25 a	°	**	***
3-Methyl-1-butanol, mg/L	201.32 cd	342.41 a	225.54 c	369.55 a	185.38 d	296.77 b	°	***	***
2-Methyl-1-butanol, mg/L	61.78 d	95.03 b	78.16 c	110.39 a	69.21 d	92.21 b	°	***	***
3-Methylbutyl acetate, μg/L	39.96 d	136.98 a	32.10 e	116.76 b	28.23 e	107.68 c	°	***	***
2-Methylbutyl acetate, μg/L	4.48 c	14.28 a	4.07 c	13.67 a	3.51 c	10.59 b	°	***	***
Isoamyl propionate, μg/L	0.19 cd	0.66 b	0.24 cd	0.86 a	0.12 d	0.29 c	°	***	***
2-Phenylethanol, mg/L	146.74 e	307.17 c	255.92 d	434.81 a	166.61 e	379.34 b	°	***	***
Phenethyl acetate, μg/L	25.24 c	51.29 b	28.53 c	56.68 a	7.36 e	13.34 d	***	*	***
Carbonyl compounds, mg/L	0.75 a	0.67 b	0.08 e	0.07 e	0.34 d	0.53 c	***	°	***
Esters, mg/L	4.04 c	9.18 a	3.76 c	8.23 b	4.03 c	8.87 a	°	***	***
Higher alcohols, mg/L	479.85 d	837.45 b	649.33 c	1018.79 a	500.50 d	856.80 b	°	***	***

^a–e Mean values in the rows marked by different letters are significantly different (two-way ANOVA, p < 0.05); ¹ significance codes: 0 < *** < 0.001 < ** < 0.01 < * < 0.05 < ° < 1.

present selected results of GC analysis of fermented molasses worts and rye mash, crucial for evaluating the distillery potency of S. boulardii yeast. A full set of analysis results is included in the Supplementary Materials.

The results of the two-factor analysis of variance (ANOVA) show that both extract content and yeast breed had a significant effect on the concentration of volatile compounds in the attenuated molasses wort, with a relationship for the second independent variable tested, i.e., extract content, for only 6 of the 31 compounds determined. In addition, the interaction between the studied variables proved significant for most of the determined compounds. The results of variance analysis gave rise to Tukey’s multiple comparisons test to show which groups differed significantly from each other. The results of the quantitative analysis of volatile compounds and the results of the statistical analysis are presented in Table 7. Over half of the identified compounds were esters, mainly ethyl esters of acetic acid and higher fatty acids. Quantitatively, on the other hand, the highest concentrations were higher alcohols, whose total concentration exceeded that of esters by up to 10 times and ranged from 145.8 mg/L (SB yeast, 10% (w/w) extract) to 405.6 mg/L (TD yeast, 10% (w/w) extract).

A higher extract content of 17% (w/w) led to higher concentrations of volatile compounds, compared to a lower extract content of 10% (w/w), but this relationship only applied to selected compounds and depended on the yeast strain used for fermentation. When analysing the total ester content, which ranged from 14.7 to 48.7 mg/L, the effect of the higher extract on the increase in ester concentration was evident for SB and TD yeast and concerned the ethyl esters of formic, acetic, butyric, isobutyric, and valeric acids. In contrast, an inverse relationship could be observed for the other esters, where higher concentrations of 3-methylbutyl acetate, 2-methylbutyl acetate, 2-phenylethyl acetate, and ethyl octanoate were recorded in the lower-extract molasses wort fermented by SB yeast.

Also, the quantitative profile of volatile compounds in worts fermented by ER yeast showed variation according to extract content, with smaller differences than for SB and TD yeasts. Of the 30 compounds determined in these samples, only 8 compounds were different, namely, 3-methylbutanal, propyl acetate, 1,1-diethoxyethane, ethyl butyrate, ethyl isobutyrate, 2-ethylhexanol, phenylacetaldehyde, and ethyl nonanoate. No statistically significant differences were found for the remaining compounds.

Of the higher alcohols determined, amyl alcohols, i.e., 3-methyl-1-butanol and 2-methyl-1-butanol, had the highest proportion (60.70–72.10%), while 2-methyl-1-propanol and 2-phenylethanol accounted for 19.20–24.67% and 7.35–14.82%, respectively. TD yeast showed a high aptitude for synthesising higher alcohols. Irrespective of the extract content, the highest concentrations of isobutyl, isoamyl, and amyl alcohols were determined in worts fermented by this yeast strain compared to worts fermented by the other two yeast strains. 1-Hexanol was determined only in wort samples fermented by TD yeast.

Concentrations of carbonyl compounds ranged from 5.56 to 25.14 mg/L, and analogous to the higher alcohols, worts fermented by TD yeast had the highest contents, including isobutyric aldehyde, 1,1-diethoxyethane, 2-methylbutanal, 3-methylbutanal, and phenylacetaldehyde.

Analysing the effect of extract content on the concentration of higher alcohols and carbonyl compounds, it can be concluded that only in the case of acetal diethyl acetaldehyde did this parameter have a similar effect during the fermentation of molasses worts by all yeast strains tested. Indeed, acetal concentration was higher in worts with an initial extract of 17% (w/w). Regarding the other compounds, only in the case of some yeast strains did their concentrations differ depending on the extract content. This was the case with SB yeast, as the concentrations of compounds such as 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isobutyric aldehyde, and diacetyl were lower in worts with an initial extract content of 10% (w/w) compared to their concentrations in worts with a higher extract of 17% (w/w).

As the extract increased, the concentration of substrates used in yeast metabolic processes during ethanol fermentation, including sugars and amino acids, increased. This may have resulted in increased production of volatile compounds, but this is an individual yeast strain characteristic. The response to a higher extract is a higher ethanol concentration, and this may translate into an increase in ethyl esters, which was confirmed in worts fermented by SB and TD yeasts. Lu Y. et al. [42], investigating the effect of sugar concentration on the composition of mango wine, showed that fermentation of a substrate with a higher sugar content increased the concentration of isoamyl acetate, reasoning that this may have been influenced by the higher concentration of isoamyl alcohol. In our study, no such relationship was found for both isoamyl acetate and 2-methylbutyl acetate and 2-phenylethyl acetate since in worts (17% w/w) with higher concentrations of higher alcohols—the precursors of these esters—their concentrations were lower compared to the composition of worts with a lower extraction (10% w/w).

In contrast to the results from the fermentation of molasses worts, the analysis of variance of the results obtained after fermentation of rye mash (Table 8) showed a significant effect of the extract on the concentration of higher alcohols, including those most critical in terms of quantity, i.e., isobutyl alcohol, isoamyl alcohol, amyl alcohol, and 2-phenylethanol. Indeed, higher concentrations of the above alcohols were determined in the higher-extract mash for all three tested yeast strains. Rye grains are rich in amino acids—precursors for the synthesis of higher alcohols in the pathway proposed by Ehrlich [43] in which the deamination of amino acids and decarboxylation of the resulting α-keto acids produce the corresponding aldehydes, which in turn are reduced to the appropriate alcohols. Compared to molasses worts, the proportion of amyl alcohols in the total sum of higher alcohols was lower, ranging from 45.40 to 52.24% and decreasing in favour of 2-phenylethanol, whose proportion was higher, ranging from 33.29 to 44.27%. A consequence of the higher concentrations of the above-mentioned higher alcohols in the mash with a higher initial extract of 17% (w/w) was also the higher concentrations of the acetate esters of these alcohols, i.e., 3-methylbutyl acetate, 2-methylbutyl acetate, and 2-phenylethyl acetate, as well as the propionic acid ester of isoamyl alcohol, compared to the mash with a lower initial extract of 10% (w/w). Fermentation of the more extractable rye mash also yielded higher ethanol concentrations, resulting in a significant effect of the extract on the increase in the major ethyl esters, i.e., ethyl acetate and ethyl formate, as well as the minor esters, i.e., ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl valerate, and ethyl hexanoate. The statistical analysis also demonstrated the yeast strain’s effect on certain esters’ concentrations. The mash fermented by SB yeast had higher concentrations of most of the esters determined. This group of compounds is one of the most important in shaping the aroma of alcoholic beverages, and even small changes in their concentration can affect the aroma of the finished product [44]. In addition, higher concentrations of diacetyl were determined in mash fermented by SB yeast compared to TD and ER yeasts. Diacetyl is formed by valine synthesis and highly depends on yeast strain and fermentation conditions. Its concentration significantly affects the aroma of alcoholic beverages due to its pronounced buttery notes [45]. In contrast, 1,1-diethoxyethane was present in the highest concentration in mash fermented by ER yeast.

3.3. Organoleptic Analysis of Rye Distillates

The results of the parametric evaluation of the distillates are summarised in

Table 9. Organoleptic parameters of rye distillates were obtained after fermentation with the ER, TD, and SB strains.

	Colour	Clarity	Aroma	Taste	Sum	Overall Taste and Aroma Impression
Yeast	[pts.]	[pts.]	[pts.]	[pts.]	[pts.]	[pts.]
SB	1.9 ± 0.1	1.9 ± 0.1	2.9 ± 1.1	10.1 ± 1.7	16.8	7.3 ± 2.4
TD	1.8 ± 0.2	1.6 ± 0.4	3.1 ± 0.5	7.7 ± 2.6	14.4	6.1 ± 2.0
ER	2.0 ± 0.0	2.0 ± 0.0	3.0 ± 0.8	8.7 ± 2.9	15.7	6.4 ± 1.5

pts.—Points.

.

In the colour category, the distillate for which the ER yeast strain was used received the maximum score from all respondents. However, the other two distillates scored similarly after considering the standard deviation. Also, the assessment of the clarity of the distillates resulted in a maximum score from all respondents for the distillate created with the ER yeast strain. The other two distillates received similar scores at 1.9 ± 0.1 points for strain SB and 1.6 ± 0.4 points for strain TD, respectively. The testers did not award any distillates that obtained the maximum number of points in the aroma category. All the distillates obtained received similar scores, with a difference of only 0.2 points between the best- and the worst-scoring samples. The most significant recorded differences in scores were in the taste category. The highest rating was given to the distillate after fermentation by SB yeast (10.1 points ± 1.7). The evaluation of the overall taste and aroma impressions indicates that the evaluators would most like to return to the distillate from the probiotic yeast strain. The other two samples received similar scores. The evaluation of the taste and aroma characteristics suggests that probiotic yeast could be used to produce spirit beverages, characterised by their specific mild taste and aroma. Despite the lack of data on spirit beverages made with Saccharomyces boulardii yeast, the literature provides information on the sensory analysis of other beverages obtained with this yeast. Díaz and collaborators [8] described the sensory evaluation results of beers obtained with probiotic yeast. These beers were judged to be similar to those obtained with other yeasts. According to the authors, the SB yeast induced a cereal character to the evaluated beverage. The researchers also noted that in the samples created with probiotic yeast, no characteristics negatively influencing the perception of the beer were detected. This yeast showed the specific character of the fermentation by-products formed, which translated into the favourable taste qualities of the beer made with SB yeast. Senkarcinova et al. [46] report that using probiotic yeast for low-alcoholic beer provides acceptable sensory attributes for the product. Others also mention that S. boulardii has no adverse effect on the aroma of alcoholic drinks [9,47].

Our results and literature data suggest that further research should be conducted to extend the potential applications of S. boulardii yeast for producing alcoholic beverages.

4. Summary and Conclusions

The research described in this article evaluates the ability of the probiotic yeast S. boulardii to ferment typical media used in distilleries. In addition to the fermentation of molasses worts, the study also assessed the fermentation of mash made from rye, which is one of the basic raw materials used in European distilleries to obtain spirit beverages. The rye mash was prepared according to the well-known “pressureless starch liberation” technology in the distillery, which is based on temperature treatment combined with the use of hydrolysis with amylolytic enzymes. Analogous experiments were carried out using the distillers’ yeasts Ethanol Red and Thermosacc Dry for comparison. The probiotic yeast showed a longer adaptation phase to fermentation and did not ferment fructose. However, the final ethanol concentrations did not differ from those obtained for the distillery strains. Analysis of volatile compounds in the fermented medium displayed statistically significant differences in the concentrations of some compounds compared to the distillery strains. Rye distillates obtained after fermentation by probiotic yeast were characterised by slightly higher overall taste and aroma sensations than rye distillates for which distillery yeasts were used. Such distillates may exhibit unique sensory characteristics desired by some consumers. The results obtained are promising for the potential production of niche distilled spirits using S. boulardii yeast. In the authors’ opinion, continuing studies on a semi-industrial or industrial scale seem intriguing to assess whether the specific characteristics of the probiotic strain will require adjustment of technological parameters for efficient processing of starchy raw materials into spirits for food purposes.